In mammals, male sex determination is initiated by SRY (sex-determining region of the Y chromosome) gene expression and followed by testicular development. This study describes specific down-regulation of the human SRY gene transcription by cAMP stimulation using reverse transcription-polymerase chain reaction experiments. Using transfection experiments, conserved nuclear hormone receptor (NHR1) and Sp1 consensus binding sites were identified as essential for this cAMP transcriptional response. Steroidogenic factor-1 (SF-1), a component of the sex-determination cascade, binds specifically to the NHR1 site and activates the SRY promoter. Activation of SF-1 was abolished by cAMP pretreatment of the cells, suggesting a possible effect of cAMP on the SF-1 protein itself. Indeed, human SF-1 protein contains at least two in vitro cAMP-dependent protein kinase (PKA) phosphorylation sites, leading after phosphorylation to a modification of both DNA-binding activity and interaction with general transcription factors such as Sp1. Taken together, these data suggest that cAMP responsiveness of human SRY promoter involves both SF-1 and Sp1 sites and could act via PKA phosphorylation of the SF-1 protein itself.