Background: Interoception refers to the central nervous system processes through which individuals become aware of their visceral feelings and internal body state. The interoceptive brain network, comprised of the insula, striatum, cingulate, and prefrontal cortex serves a homeostatic purpose, integrating internal and external signals with cognition, affect, and motivation to guide the regulation of body physiology. Substance use disorder (SUD) may be a manifestation of chronic, maladaptive attempts to regulate homeostasis via alcohol and other drug use. Yet little is known about how dysregulated interoception develops and whether interoceptive alteration precedes or is a consequence of heavy substance use. This project begins to address these questions with a series of three neuroimaging studies examining the functional and structural neural underpinnings of interoception in adolescent substance users. Methods: Adolescents ages 15-17 who met diagnostic criteria for SUD (SUD+ group), adolescents who had experimented with substances but did not meet criteria for SUD (EXP group), and non-using control adolescents (CON group) completed a clinical interview, questionnaires, structural neuroimaging, and two functional magnetic resonance imaging (fMRI) paradigms designed to induce pleasant and aversive interoceptive sensations. In the pleasant paradigm, adolescents experienced a soft brush stroke to the skin (17 SUD+, 19 EXP, 19 CON); during the aversive paradigm, adolescents were subjected to an inspiratory breathing load (17 SUD+, 15 EXP, 19 CON). fMRI analyses examined group differences in blood oxygen level dependent (BOLD) signal change with linear mixed effects models. Gray matter volume, cortical surface area, and cortical thickness of key interoceptive regions were estimated using T1- weighted structural data (24 SUD+, 28 EXP, 23 CON) and group differences were examined via ANOVA. Regression analyses were used to probe the relationship of substance use, clinical symptoms, and subject characteristics to neural structure and function of the insular cortex. Results: Group differences were observed in BOLD response contrast within the interoceptive network (ps<.05), despite comparable task performance, similar levels of body awareness, and similar ratings of the pleasant and aversive sensations. During pleasant stimulation, SUD+ and EXP showed less BOLD response in posterior insula (PIC), anterior cingulate (ACC), and numerous frontal cortical regions compared to CON. SUD+ also demonstrated heightened BOLD response in the lentiform nucleus and had the greatest activation change from anticipation to stimulation in the dorsal anterior insula (AIC). During aversive stimulation, both SUD+ and EXP had exaggerated activation patterns in the dorsal AIC, but only the SUD+ group showed a significantly elevated response relative to CON in the PIC. By contrast, the rostral AIC was sensitive to anticipating the aversive stimulus in the CON group but deactivated during this condition in the SUD+ group. In the frontal cortex, SUD+ had elevated activation to the aversive stimulus in the left ACC, yet blunted reactivity in the left middle frontal gyrus and right rostral ACC. Across all subjects, higher levels of sensation seeking were associated with decreased insula activation during aversive loaded breathing (p=.01) Groups did not differ on any brain volumetric, surface area, or cortical thickness variable examined (ps>.05) Conclusions: Findings indicate that adolescent substance use history is associated with aberrant brain response patterns of interoceptive processing in the absence of group differences in subjective interoceptive awareness or gray matter morphology. The nature of this functional dysregulation depends on the valence of the physiological experience. That aberrant activation patterns were seen in youth with SUD, but also in adolescents with sub-clinical substance exposure advances the theory that differences in the perception and processing of visceral sensations may be, at least in part an early predisposing factor for substance misuse, rather than a consequence of addiction or long-term substance exposure. By further elucidating a novel mechanism that may contribute to addiction risk, this study may aid in the development of innovative interventions to improve visceral processing.