The chronic effect of CAMP‐dependent regulation on adrenocortical steroidogenesis is known to be revealed in the stimulation of the biosynthesis of steroidogenic enzymes. P‐450(SCC), one of the enzymes, catalyzes the first and the rate‐limiting reaction in steroidogenesis from cholesterol and its synthesis is regulated by CAMP. In order to investigate cis‐acting DNA elements of this gene in response to CAMP‐dependent regulation, we have constructed a fusion gene (pSCC5.4k) by ligating the 5′‐flanking and the upstream untranslated region (5.4 kb) of the human P‐450(SCC) gene to the structural gene for chloramphenicol acetyltransferase (CAT) and transfected it into various culture cells including Y‐1 (mouse adrenal tumor), L929 (mouse fibroblast), HTC (rat hepatoma) and Hepa‐1 (mouse hepatoma). Only Y‐1 cells transfected with pSCC5.4k were found to express transiently the enhanced CAT activity in response to the cAMP analogue, cyclic dibutyryl‐AMP (Bt2cAMP). Primer‐extension analysis of RNA prepared from the cells treated with or without Bt2cAMP showed that the enhanced CAT activity was due to an increase in the CAT mRNA and that the transcription start site, determined here with the human P‐450 gene in the adrenal cortex, was correctly utilized with the fusion gene in the transient expression system. Forskolin and cholera toxin, activators of adenylate cyclase, also increased the expression of the CAT activity in the Y‐1 cells. It has been demonstrated, therefore, that the CAMP‐dependent regulation of the P‐450(SCC) gene in adrenal cortex is faithfully reflected in the transient expression system using Y‐1 cells and the fusion gene and that a cis‐acting DNA element(s) in response to cAMP is present within the 5′‐flanking sequence (5.4 kb) of the P‐45O(SCC) gene.