AbstractCYP11A1encodes the first enzyme of steroid biosynthesis, cytochrome P450scc. The expression ofCYP11A1in the nervous system allows neurosteroids to be synthesized de novo. In the classic steroidogenic tissues, adrenals and gonads, the key regulator controllingCYP11A1expression is steroidogenic factor‐1 (SF‐1), but the transcriptional regulation ofCYP11A1in the brain is unclear. We recently used the 4.4‐kb regulatory region of the humanCYP11A1gene to drive Cre recombinase expression in the diencephalon and midbrain. In this study, we characterized the regional‐specific expression of Cre reporter in the SCC‐Cre transgenic brain using a transient Cre/ROSA26R transgenic system. Mutation of either the upstream or proximal SF‐1 binding site did not affect brainCYP11A1promoter activity. The upstream SF‐1 binding site, however, is required forCYP11A1promoter function in the embryonic adrenals. The 3.8‐kb promoter, like the 4.4‐kb length promoter, directed Cre expression in the diencephalon, midbrain and olfactory epithelium, whereas Cre expression controlled by the 2.7‐kb promoter was only observed in the caudal part of midbrain. This suggests that the 5′‐flanking region between 3.8 and 2.7 kb contains a crucial element for activation ofCYP11A1promoter in the diencephalon, olfactory epithelium and the anterior part of midbrain. Thus we have identified regions of the promoter that controlCYP11A1expression in the brain and embryonic adrenals. J. Cell. Physiol. 226: 1998–2005, 2011. © 2010 Wiley‐Liss, Inc.