Transcription of the StAR gene has been shown to be positively regulated by cAMP via steroidogenic factor-1. PGF2α, working through protein kinase C, may act indirectly, reducing cAMP-mediated StAR gene expression, as well as directly, inhibiting transcription of the StAR gene via binding of negative regulatory transcription factors. Pharmacologic activation of the PKC signal transduction pathway by the phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), has been shown to stimulate transcription of genes that are regulated by the transcription factor complex AP-1. The AP-1 complex consists of cJun homodimers and heterodimers of cJun and cFos family members. cJun/cFos heterodimers have been shows to bind TPA response elements (TREs) following phorbol ester stimulation. Recently, however, cFos was shown to have a dominant negative effect on gene transcription when expressed at levels which exceed those of cJun. To determine whether cFos could regulate transcription of the StAR gene, the rat StAR promoter was examined for potential TRE regulatory motifs. Two putative TREs at positions -272 (proximal = pTRE) and -1643 (distal = dTRE), relative to the StAR initiation codon, were identified. Electrophoretic mobility shift analysis, incubating nuclear extracts with probes corresponding to the pTRE and dTRE regulatory motifs, demonstrated binding to these putative regulatory elements with high affinity. The binding of nuclear factors to the pTRE was abrogated upon addition of cFos antiserum whereas binding to the distal site was supershifted upon addition of cFos antiserum. Addition of cJun antiserum had no effect on nuclear factor binding to either TRE. Luciferase assays were conducted with both a 450 bp minimal StAR promoter construct, pGL3-mStAR, containing only the pTRE, and a 2.2 kb StAR promoter construct, containing both TREs (pGL3-StAR). Adrenal Y1 tumor cells were transfected with pGL3-mStAR or pGL3-StAR in the presence or absence of 1 mM cAMP. Luciferase activity in cells transfected with the StAR promoter constructs alone were significantly increased (2-fold) in response to the addition of cAMP (p< 0.05). Co-transfection of the expression plasmid for cFos with the StAR promoter constructs caused a decrease in both basal and cAMP-stimulated StAR promoter-linked luciferase activity. Co-transfection of cFos caused a 55% and 36% reduction in basal luciferase activity (p< 0.05) in the 450 bp and the 2.2 kb StAR promoter constructs, respectively. Overexpression of cFos also caused a 61% and 49% reduction in cAMP stimulated luciferase activity (p < 0.05) in these respective constructs. The results of this study provide the first evidence that cFos represses transcription of the StAR gene and limits cAMP responsiveness of the rat StAR promoter.