Recent studies have shown that corticotropin releasing factor (CRF) stimulates c-fos gene expression in the AtT-20 corticotroph cell line, and that overexpression of c-Fos results in activation of POMC gene transcription. Since transactivation by c-Fos requires dimerization with a Jun family member to form the active transcription factor AP-1, we have examined the expression of multiple fos and jun related genes and have correlated their expression with AP-1 DNA binding activity in AtT-20 nuclear extracts after stimulation with CRF. Although basal expression of c-fos mRNA was extremely low, it was rapidly and transiently stimulated in AtT-20 cells following administration of either constant or a single pulse of CRF. In contrast, basal expression of c-jun mRNA was slightly higher and underwent little or no change in response to CRF. Specific ribonuclease protection analysis showed that in addition to c-fos, mRNA transcripts encoding fos B and jun B were rapidly stimulated in response to CRF, though levels of induced fos B mRNA were 20-40 times lower than c-fos or jun B, respectively. Gel shift analysis demonstrated that CRF caused a sustained increase in AP-1 DNA binding to both a canonical AP-1 element as well as to the POMC exon-1 AP-1 site. Studies with specific antisera directed against c-Fos revealed that although no c-Fos could be detected in AP-1 complexes in basal cell extracts, c-Fos became a prominent component of AP-1 following CRF stimulation, reaching maximal levels by 4 h. Despite the fact that AP-1 DNA binding activity remained elevated for at least 24 h after CRF, c-Fos was most prominent during the early phase of the response. Similarly, JunB was shown to be a major component of AP-1 DNA binding activity in CRF-stimulated AtT-20 nuclear extracts that persisted for at least 24h after stimulation. Despite the obvious induction of fos B mRNA in response to CRF, FosB protein was not detected in DNA bound AP-1 complexes. These data demonstrate that CRF is a potent stimulus for corticotroph expression of c-fos, jun B and fos B, and suggest that the subsequent increase in AP-1 may play a role in activation of gene expression and/or as a modulator of glucocorticoid receptor function.