The 3alpha-hydroxy,5alpha-reduced pregnane steroids, allopregnanolone and allotetrahydrodeoxycorticosterone, are the most potent endogenous positive modulators of GABA(A) receptor-mediated inhibition. This study presents the first immunohistochemical examination of the cellular distribution of 3alpha-hydroxy,5alpha-reduced pregnane steroids across the brain. We found a widespread distribution in the adult rat, with dense immunolabelling in the olfactory bulb, striatum and cerebral cortex, and lower density labelling in the brainstem reticular formation. In general terms, this distribution accords with the regional concentrations of 3alpha-hydroxy,5alpha-reduced steroids determined, in other laboratories, by brain region sampling and either gas chromatography-mass fragmentography or radioimmunoassay. However, immunohistochemistry allowed for a more detailed examination of regional distribution and cellular specificity. All immunoreactivity was confined to the cell bodies and thick dendrites of neurones; no identifiable glia were labelled. In most brain areas, the location and morphology of labelled cells identified them as excitatory neurones. In addition, cell populations known to be projecting GABAergic neurones (e.g. cerebellar Purkinje cells) were immunoreactive, whereas local inhibitory neurones generally were not. The cellular distribution of 3alpha-hydroxy,5alpha-reduced steroids suggests that sensory, motor, limbic and homeostatic systems can be influenced by neurosteroids at multiple stages of processing.