Regulation of steroidogenesis in classic endocrine tissues is mediated by transcriptional regulation of the P450scc gene, which encodes the first and rate-limiting cholesterol side-chain cleavage enzyme. We previously showed that P450scc messenger RNA is regionally expressed in the adult rat brain, primary glial cultures, and C6 glioma cells. Expression of P450scc in the brain results in the de novo synthesis of neurosteroids, a class of steroid hormones that are active at gamma-aminobutyric acidA and N-methyl-D-aspartate receptors. We determined whether P450scc expression is transcriptionally regulated in neural cells, using the same DNA sequences and nuclear proteins as classic steroidogenic adrenal and Leydig cells. The transcriptional activity of deletional mutants of 2.5 kilobases of the 5'-flanking regulatory region of the rat P450scc gene cloned into a luciferase reporter gene was assessed in mouse adrenocortical Y-1, mouse Leydig MA-10, rat C6 glioma, rat GC somatotrope, and mouse GT1-7 neurosecretory cell lines. P450scc was transcriptionally regulated in Y-1, MA-10, and C6 glioma cells, but not in GC or GT1-7 cells. In one region (-94/-35), putative steroidogenic factor-1-binding sites appeared to be critical for the basal transcriptional activity and cAMP responsiveness in steroidogenic Y-1 and MA-10 cells, but had no function in rat C6 cells. DNA sequences between -94/-130 mediated both basal and cAMP-inducible transcriptional activity in C6 cells. Gel mobility shift assays showed that one nuclear protein binding to DNA sequences between -54 and -35 was abundant in MA-10 and Y-1 cells, but was absent from C6 cells, whereas another nuclear protein, binding to DNA sequences between -94 and -130 was abundant in C6 cells, but was rare in MA-10 cells and absent from Y-1 and other adrenocortical cells. Although the DNA sequence between -94 and -130 contains an Sp1 site, Sp1 did not bind to this site. Nevertheless, this GC-rich region was critical for nuclear protein binding and for basal and cAMP-induced transcriptional regulation in both C6 and MA-10 cells. These observations demonstrate that the rat P450scc gene is transcriptionally regulated in glioma cells, but its regulation in glial cells involves a DNA element different from those used in classic steroidogenic tissues. The results further suggest that steroidogenic factor-1 is not involved in regulating neurosteroidogenesis.