Cultured rat and ovine anterior pituitary cells were treated with a range of doses (0.01–1,000 n M) of arginine vasopressin (AVP) and ovine corticotropin-releasing factor (CRF), alone or in combination, and medium and cell content of immunoreactive (ir-)ACTH determined. In rat cells, a dose-response curve to CRF was obtained, with a threshold dose of 0.1 n M; AVP was much less effective alone, but augmented CRF responses when administered with CRF. In ovine pituitary cells AVP markedly stimulated ACTH release in a dose-dependent fashion, and with a threshold of 0.1 n M; in contrast, CRF increased ACTH release over basal only at doses > 100 n M. In combination, subthreshold doses of AVP potentiated rat pituitary cell responses to CRF; addition of 1 n M of AVP to varying doses of CRF was more effective in terms of ACTH release than addition of 1 n M of CRF to increasing doses of AVP. In contrast, in ovine cells the addition of 1 n M CRF to increasing doses of AVP elicited a larger ACTH response than the addition of 1 n M AVP to increasing doses of CRF. Dexamethasone pretreatment (5 n M) for 48 h significantly decreased CRF potentiation of AVP-stimulated ACTH release in ovine cells. These studies confirm that CRF is a more potent stimulus of ACTH release than AVP in the rat, and establish that in contrast AVP is a much more potent stimulus of ACTH secretion than CRF in isolated ovine pituitary cells.