Carbonyl sulfide (COS), a high-priority Clean Air Act chemical, was evaluated for neurotoxicity in short-term studies. F344 rats were exposed to 75-600 ppm COS 6 h per day, 5 days per week for up to 12 weeks. In rats exposed to 500 or 600 ppm for up to 4 days, malacia and microgliosis were detected in numerous neuroanatomical regions of the brain by conventional optical microscopy and magnetic resonance microscopy (MRM). After a 2-week exposure to 400 ppm, rats were evaluated using a functional observational battery. Slight gait abnormality was detected in 50% of the rats and hypotonia was present in all rats exposed to COS. Decreases in motor activity, and forelimb and hindlimb grip strength were also detected. In rats exposed to 400 ppm for 12 weeks, predominant lesions were in the parietal cortex area 1 (necrosis) and posterior colliculus (neuronal loss, microgliosis, hemorrhage), and occasional necrosis was present in the putamen, thalamus, and anterior olivary nucleus. Carbonyl sulfide specifically targeted the auditory system including the olivary nucleus, nucleus of the lateral lemniscus, and posterior colliculus. Consistent with these findings were alterations in the amplitude of the brainstem auditory evoked responses (BAER) for peaks N3, P4, N4, and N5 that represented changes in auditory transmission between the anterior olivary nucleus to the medial geniculate nucleus in animals after exposure for 2 weeks to 400 ppm COS. A concentration-related decrease in cytochrome oxidase activity was detected in the posterior colliculus and parietal cortex of exposed rats as early as 3 weeks. Cytochrome oxidase activity was significantly decreased at COS concentrations that did not cause detectable lesions, suggesting that disruption of the mitochondrial respiratory chain may precede these brain lesions. Our studies demonstrate that this environmental air contaminant has the potential to cause a wide spectrum of brain lesions that are dependent on the degree and duration of exposure.