Methyl tert-butyl ether (MTBE) is a volatile organic compound that can function as a fuel oxygenate, increasing the efficiency of combustion of gasoline. Its use in this capacity was mandated in the 1990s, during winter months, in areas of the U.S. where ozone and carbon monoxide levels exceeded EPA limits. Though it has been discontinued as a fuel additive in the US, its widespread previous use has resulted in contamination of ground and surface waters, mainly from leaking underground storage tanks. It has proven to be a persistent organic pollutant, and is resistant to remediation. While the acute toxicity of MTBE is low, its chronic health effects in humans are largely unknown, thus accurate risk assessment is difficult. Cancer bioassays have shown increased testicular tumors in male rats and increased hepatic tumors in female mice, suggesting that MTBE may act as an endocrine disruptor. MTBE does not appear to be genotoxic, its proposed mechanism is a mitogenic effect caused by disruption of the hypothalamic-pituitary-gonadal control axis. MTBE has been shown to have no effect on fertility in male and female rats. Previous studies have demonstrated dose-dependent reduction in serum testosterone in male rats following MTBE exposure, and reduced testosterone production by rat testicular interstitial cells, in cell culture, when incubated with MTBE. One previous study has shown a significant reduction in testosterone concentration in testicular interstitial fluid in Sprague-Dawley rats dosed at 1500 mg/kg/day for 14 days, a finding that appears to be at odds with the demonstrated lack of fertility impairment. The objectives of this study were to attempt to replicate previous dose-dependent reductions in serum testosterone in male rats, and to examine the effects of MTBE in testosterone levels within the testis. Exposure levels were selected to parallel those used in a previous cancer bioassay. Juvenile male Sprague-Dawley rats, n=10 per dose group, were exposed to MTBE in corn oil vehicle by gavage at 0, 600, or 1200 mg/kg/day for 14 days. Additional animals, n=6 per dose group, were treated identically to the core groups, but each received an intraperitoneal injection of hCG, 50 IU/kg, 2.5 hours prior to sacrifice. Three unscheduled deaths occurred in the high dose group. MTBE treatment caused dosedependent increases in adrenal weight and decreases in rate of body weight gain. There was no significant reduction in serum testosterone with MTBE treatment, in the absence of hCG stimulation. hCG produced significant increases in serum and intratesticular testosterone in all groups. In the 600 mg/kg/day MTBE treated animals who received hCG there was a significant reduction in the amplitude of this response when measured by radioimmune assay, but this effect was equivocal when measured by ELISA. There were no dose-effects of MTBE on intratesticular testosterone in any group. These findings tend to support general toxicity rather than a specific mechanism of MTBE targeting the HPT axis.