Neurosteroids are steroids that are made in and act on neural tissue in an autocrine/paracrine fashion. Steroidogenesis in classical endocrine tissues (e.g., adrenal, gonads, placenta) is initiated by conversion of cholesterol (Chol) to pregnenolone (Preg) by the mitochondrial cholesterol side chain cleavage enzyme, P450scc. Rat neonatal forebrain cultures can mimic adrenal Steroidogenesis by converting 3H mevalonolactone, a precursor of Choi, to 3H Choi, 3H Preg, and 3H 20-OH Preg. To determine whether P450scc mediates this conversion, we analyzed RNA from rat brains, primary cultures of glial cells, and C6 glioma cells for P450scc mRNA by RNase protection assays, but found none. However P450c11β (11β hydroxylase), but not P450c11AS (aldosterone synthase) nor P450cl7 (17α hydroxylase) was detected. Only by RT/PCR analysis could we detect P450scc mRNA, but not P450cl7 nor P450c11AS mRNAs. P450scc mRNA is most abundant in the cortex, but is also found in the amygdala, hippocampus and midbrain of both male and female rats. P450c11β mRNA, also, is found mainly in the cortex of male and female rats, but is more abundant in the female than male hippocampus. Purification of our mixed primary glial cultures shows that Type-1 astrocytes synthesize P450scc but not P450c11β mRNA. Western blotting and immunocytochemistry show that P450scc protein is almost as abundant in our cultures as in Y-1 cells, while P450scc mRNA is orders of magnitude less abundant suggesting that the protein is very stable in the brain. Thus the synthesis of classic steroids (glucocorticoids and progestins) and neurosteroids (allopregnanolone) can be regulated in situ in the brain, and do not require extra-neural substrates.