The rat steroid cytochrome P450 17 alpha-hydroxylase/c17-20 lyase (rP450c17) gene is transcriptionally regulated in steroidogenic tissues. Previous studies showed that one DNA element located between -75 and -50 base pairs (bp) upstream from the transcriptional initiation site mediated both the basal and cAMP-regulated transcription of rP450c17. Using a series of mutant oligonucleotides in gel mobility shift assays and in functional assays, it is now shown that a core sequence of 12 bp, located at -58/-69 bp, is essential for nuclear protein binding and transcriptional activation. Mutant oligonucleotides cloned into a luciferase reporter gene construct containing a heterologous thymidine kinase promoter, transfected into mouse Leydig MA-10 and adrenocortical Y-1 cells, gave results consistent with those of gel shift assays. Mutants that abolished binding of the nuclear protein to DNA abolished the basal transcription of the gene as well as the responsiveness to cAMP, whereas those mutants that did not abolish binding of the nuclear protein to DNA still showed strong basal transcription as well as responsiveness to cAMP. Comparison of the binding sequence with the consensus binding site for the orphan nuclear receptor steroidogenic factor-1 (SF-1) showed that eight of nine bases were identical. However, the sequence from rP450c17 includes an additional three bases at the 5'-end, not previously demonstrated to be important for SF-1 binding. Recombinant rat SF-1 protein expressed in Escherichia coli binds to this sequence, and antibodies raised against rat SF-1 abolish binding of both recombinant SF-1 and the nuclear protein from Y-1 and MA-10 cells. These observations demonstrate that this region of the rP450c17 gene is responsible for both the basal transcription and cAMP inducibility and is bound by the orphan nuclear receptor SF-1. It is further shown that SF-1 can be phosphorylated in vitro by protein kinase A. This phosphorylation occurs at serine and threonine residues and results in decreased binding to the rP450c17 -58/-69 element. Since SF-1 mediates cAMP-induced transcriptional regulation of the rat P450c17 gene, phosphorylation of SF-1 via protein kinase A is likely to play a regulatory role in transcriptional activation.