Synthetic ovine corticotropin releasing factor (CRF) at 0.03–30 ng/ml evoked a significant linear release of ACTH in pituitary monolayer cell cultures. Arginine vasopressin (AVP) at 3–30 ng/ml induced a slight ACTH release in the same culture system. CRF at 100 ng/ml greatly increased the medium ACTH with intracellular ACTH remaining relatively constant. In perifused pituitary quarters, both AVP and CRF evoked significant release of ACTH. When AVP was added in combination with synthetic ovine CRF, an additive or synergistic effect was observed on the CRF-induced ACTH release in both in vitro systems. Preincubation of cultured pituitary cells with synthetic ovine CRF for 1 h did not have a significant effect on the subsequent AVP-induced ACTH release. However, when cells were preincubated for 6 h with CRF at 1 and 100 ng/ml, the AVP-induced ACTH release was significantly increased, but preincubation with AVP suppressed both the basal and AVP-induced ACTH release. These results suggest that synthetic ovine CRF stimulates not only the release of ACTH but also the synthesis of ACTH and that AVP can evoke significant ACTH release from corticotrophs which have enough releasable ACTH or substances needed for ACTH release under the influence of CRF.