AbstractmRNA for the regulatory subunit RIIb of cAMP-dependent protein kinase is stimulated more than 50-fold by cAMP in primary cultures of rat Sertoli cells. We have previously shown that this induction involves regulation of transcriptional activation as well as mRNA stabilization. The rat RIIb gene contains no cAMP response element (CRE), and the induction of RIIb mRNA is slow and requires on-going protein synthesis. When a construct containing the 5 0 -flanking region of the RIIb gene upstream of a CAT reporter was transfected into Sertoli cells by the calcium phosphate method, low and variable responses to cAMP (three-to fivefold) were observed, whereas a 15-to 20-fold increase in reporter activity by cAMP was observed after lipofectamine transfection. Interestingly, when a vector containing CRE elements upstream of a reporter gene was transfected into Sertoli cells, the responses to cAMP were similar regardless of the transfection method used. We have also demonstrated that increased intracellular levels of calcium by A23187 and thapsigargin dramatically inhibit cAMPmediated induction of RIIb mRNA, but not the mRNA for the CRE-containing RIa gene. Furthermore, decreased cAMP responsiveness of endogenous RIIbmRNA (but not RIa) was also observed in calcium phosphate-transfected Sertoli cells but not in lipofectamine-transfected cells. Thus, calcium-mediated reduction in cAMP response appears to be a gene-specific phenomenon.