To determine if neurosteroids (steroids synthesized in the brain) are produced by enzymes found in steroidogenic tissues, we determined if mRNA for five steroidogenic enzymes could be detected in brain tissues or cultured cells. We detected mRNAs for adrenodoxin, P450scc (cholesterol side-chain cleavage enzyme) and P450c11 beta (11 beta-hydroxylase) but not for P450c17 (17 alpha-hydroxylase/17,20 lyase) or P450c11AS (aldosterone synthase) in rat brains and cultures of rat glial cells. P450scc mRNA abundance in brain or primary glial cultures was approximately 0.01% of that found in the adrenal, but more P450scc mRNA was detected in C6 glial cells. Both P450scc and P450c11 beta mRNAs were most abundant in the cortex, but there were region-specific differences for both mRNAs, and sex-specific differences for P450c11 beta mRNA. P450scc mRNA was equally abundant in mixed glial cultures containing both astrocytes and oligodendrocytes as in astrocyte-enriched cultures, and P450scc immunoreactivity co-localized with GFAP immunoreactivity in cultured astrocytes. P450c11 beta mRNA was not detected in the mixed primary glial cultures for the C6 glioma cell line that synthesize P450scc mRNA, suggesting that glial cells do not synthesize P450c11 beta mRNA. Thus some of the same enzymes involved in steroidogenesis in classic endocrine tissues are found in a cell-specific and region-specific fashion in the brain. Neurosteroids may be derivatives of known classic steroids, and/or may function through non-classic steroid hormone receptors, such as GABAA, N-methyl-D-aspartate, and corticosterone receptors.