The cholesterol side-chain cleavage enzyme (P450scc) encoded by CYP11A1 gene catalyzes the conversion of cholesterol into pregneneolone, which is the first step in steroid biosynthesis and regulated by tropic hormones via cAMP-PKA pathway. The regulatory region mediating signal of cAMP on the transcription of human CYP11A1 gene contains upstream cAMP-responsive sequences (U-CRS) and proximal cAMP-responsive sequences (P-CRS). The P-CRS, located in -145 bp, harbors at least three highly conserved sequences including TATA box, SF1-binding site and Sp1-like sequence. By electrophoretic mobility shift assay, we showed oligonucleotide -117/-94 formed two specific DNA-protein complexes with nuclear extract from adrenocortical Y1, placental JEG-3, or cervical HeLa cells. The oligonucleotide containing Sp1 consensus sequence formed the same pattern as -117/-94 did. Although Sp1 was always stronger, -117/-94 and Sp1 oligonucleotides were effective competitors for each other. These data indicated a weak Sp1-binding site located in -117/-94 fragment. To test for its function, the -117/-94 fragment was fused to the TATA box of OVEC vector, before transfection into Y1 or JEG-3 cells. Its transcription activity was measured by RNA analysis after 8-Br-cAMP treatment. The results of primer extension showed -117/-94 directed cAMP-dependent transcription. Based on the observations of DNA-protein interaction assay and transcription activity assay, we conclude that a weak Sp1-binding site in the -117/-94 fragment is important for transcription of the CYP11A1 gene by interacting with basal transcription factors.