Transcription of the cytochrome P450 17 (CYP17) gene is regulated by cAMP-dependent binding of steroidogenic factor-1 (SF-1) to its promoter in the adrenal cortex. Using temporal chromatin immunoprecipitation and mammalian two-hybrid experiments, we establish the reciprocal presence of coactivators [general control nonderepressed (GCN5), cAMP response element-binding protein-binding protein, p300, p300/cAMP response element-binding protein-binding protein CBP associated factor, p160s, polypyrimidine tract associated splicing factor, and p54nrb], corepressors (class I histone deacetylases, receptor interacting protein, nuclear receptor corepressor, and Sin3A), and SWI/SNF (human homolog of yeast mating type switching/sucrose nonfermenting) and imitation SWI chromatin remodeling ATPases on the CYP17 promoter during transcription cycles in the H295R adrenocortical cell line. A ternary GCN5/SRC-1/SF-1 complex forms on the CYP17 promoter with cAMP-dependence within 30 min of cAMP stimulation, and corresponds with SWI/SNF chromatin remodeling. This complex is sensitive to the SF-1 antagonist sphingosine and results in decreased transcription of CYP17. GCN5 acetyltransferase activity and carboxy terminus binding proteins alternatively mediate disassembly of the complex. This work establishes the temporal order of cAMP-induced events on the promoter of a key steroidogenic gene during SF-1-mediated transcription.