Steroidogenic acute regulatory protein (StAR) is a 30-kDa protein involved in the transport of cholesterol to the inner mitochondrial membrane and thus plays a key role in steroid biosynthesis. To clarify the implications of this protein in neurosteroid biosynthesis, we examined the possible expression of a StAR transcript in the adult rat CNS and detected it. cDNA cloning and sequencing analysis revealed that two forms of StAR mRNAs are expressed in the brain in the same manner as in the adrenal gland, indicating that they are fully functional and not minor gene transcripts. An RNase protection assay quantitatively revealed that the amount of the rat StAR transcript in brain was two to three orders of magnitude lower than that in the adrenal gland. An in situ hybridization study, involving antisense riboprobes, revealed that StAR transcripts were abundant in the cerebral cortex, hippocampus, dentate gyrus, olfactory bulb, cerebellar granular layer, and Purkinje cells. Furthermore, other steroidogenic enzymes, side-chain cleavage cytochrome P-450SCC (CYP XIA1) and 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (EC 1.1.1.145), were found to be coexpressed in the hippocampus, dentate gyrus, cerebellar granular layer, and Purkinje cells. These findings strongly indicate that neurosteroids are synthesized in a region-specific manner in the brain.