Differences in the cellular distribution of antioxidant defense mechanisms in heterogeneous tissue such as the nervous system are likely critical determinants of differential sensitivity to toxicants. Regional and cellular localization of reduced glutathione (GSH) in central and peripheral nervous tissue was determined from the pattern of fluorescence observed in tissue sections stained with mercury orange; localization was confirmed using a novel histofluorochromatic staining method, o-phthaldialdehyde (OPT). Excellent concordance between the distribution of fluorescence obtained with mercury orange and OPT staining was observed. Depletion of GSH by treatment with diethyl maleate resulted in a diminution in both mercury orange and OPT histofluorescence. Generally, strong staining of the CNS neuropil was seen with little or no observable fluorescence in neuronal somata. The cerebellar granular cells were an exception, exhibiting fluorescence with both mercury orange and OPT. Cerebellar Purkinje cells exhibited nonuniform fluorescence with mercury orange but generally uniform staining with OPT. In contrast to the patterns observed in the CNS, the sciatic nerve and the sensory cell bodies of the lumbar dorsal root ganglia exhibited prominent fluorescence with both mercury orange and OPT. Reduced glutathione in the central nervous system appears primarily localized in the neuropil and white matter tracts; with a few exceptions, the neuronal somata do not appear to contain appreciable amounts of GSH. The heterogeneous distribution of GSH and enzymes involved in the detoxification and/or excretion of xenobiotics in the nervous system may form a basis for selective cellular and/or regional expression of neurotoxicity.