We have compared regulation of progesterone receptor (PR) gene expression in MCF-7 human breast cancer cells by cAMP and that by estradiol (E2) and insulin-like growth factor-I (IGF-I). Treatment of cells with 8-bromo-cAMP or agents known to activate protein kinase-A, namely cholera toxin plus 3-isobutyl-1-methylxanthine (CT plus IBMX; which increased intracellular cAMP > 10-fold) evoked an increase in PR protein levels as did treatment with IGF-I or E2. Increases in PR caused by IGF-I were not accompanied by increases in PR mRNA, whereas PR mRNA levels were markedly induced by E2 and CT plus IBMX, showing regulation at different levels by these agents. The increases in PR mRNA evoked by E2 or CT plus IBMX were almost completely abolished by treatment with antiestrogen. Treatment with cycloheximide inhibited CT- plus IBMX-mediated PR mRNA stimulation, but not the induction of E2, indicating that the PR mRNA response to cAMP is not a primary one and probably requires de novo protein synthesis. Distinct effects of serum were observed on the expression of PR in MCF-7 cells. PR mRNA and protein were consistently elevated when cells were cultured under low serum conditions (0-0.5% charcoal dextran-treated calf serum) and were reduced as the serum concentration was increased, suggesting that a serum factor(s) repress constitutive PR levels. Also, the ability of CT plus IBMX to stimulate PR declined markedly for cells grown in medium containing higher (5%) serum levels; by contrast, the ability of E2 to induce PR was increased at the higher serum concentration. Thus, unknown serum factors interfere with the action of cAMP in up-regulating PR, whereas serum factors are important for the effectiveness of E2 in stimulating PR. These observations indicate that regulation of PR occurs at different levels by several factors (cAMP, E2, and IGF-I) and imply that cAMP, serum factors, and growth factors, such as IGF-I, in addition to E2 will be of importance in determining PR levels and, hence, cell sensitivity to progestins.