CRF is a potent hypophysiotropic factor which stimulates POMC-producing cells in both the intermediate and anterior pituitary. Although its secretagogue effects and its stimulatory action on POMC gene expression are well documented, the mechanisms by which CRF modulates gene regulation are poorly understood. In this study we have investigated the mechanisms by which CRF stimulates the immediate early gene c-fos. Studies were performed in the corticotroph-derived AtT20 cell line. We show that CRF induces a transient increase in c-fos mRNA levels. This induction is reduced by blockade of calcium entry and by calmodulin inhibitors, suggesting that the CRF-induced c-fos increase is mediated in part by the second messenger Ca2+ and the Ca2+/calmodulin kinase. When protein kinase-A (PKA) was inhibited by introduction of a mutated regulatory subunit of PKA that lacks cAMP-binding sites, the stimulation of c-fos mRNA by CRF was abolished. Taken together, these results suggest that CRF activates the c-fos protooncogene via PKA and the Ca2+/calmodulin kinase. These results were confirmed and extended by gene transfer studies using chimera genes containing c-fos promoter sequences coupled to the chloramphenicol acetyl transferase reporter gene. This series of experiments shows that CRF stimulates c-fos transcription by mechanisms requiring PKA activation. Furthermore, cotransfection experiments with the POMC promoter linked to the chloramphenicol acetyl transferase reporter gene along with an expression vector coding for cFOS showed efficient stimulation of POMC gene transcription by cFOS. In summary, c-fos mRNA accumulation is an early genomic signal in pituitary cells in response to CRF, and cFOS may represent a signal controlling POMC gene expression.