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Corticotropin-releasing factor (CRF) has been characterized on the basis of its intrinsic activity to release corticotropin from cultured rat anterior pituitary cells. Injected in intact rats, CRF increases adrenocorticotropic hormone (ACTH) release. Endogenous CRF-like immunoreactivity was detected in the cytoplasm and nucleus of corticotrophs. Using an antirat CRF serum, a similar location of CRF-like immunoreactivity was observed in lactotrophs: cytoplasmic matrix, secretory granules, nucleus and, to a lesser degree, the plasma membrane level were stained. One injection of CRF increased the plasma ACTH concentration 4-fold after 15 min, while plasma prolactin (PRL) increased 2.7-fold 5 min after injection. In vitro, incubation of female pituitary cells with rat CRF (10^–10–10^–8M) had no significant effect on PRL secretion. In contrast, after 4 days of in vitro pretreatment with 17β-estradiol (10^–9M), rat CRF stimulated PRL secretion by 42%. In situ hybridization of whole pituitary slices showed that rat CRF injection significantly increased the labeling of corticotrophs using an ACTH-cDNA probe, but had no significant effect on the labeling of lactotrophs using a PRL riboprobe. These results indicate that CRF is a factor which can modulate PRL release but not the synthesis of PRL.