The effect of synthetic ovine CRF on ACTH secretion of dispersed, domestic fowl pituitary cells was investigated. Cells preincubated for 2 h, 16 h, or after 48-h culture were incubated briefly with CRF (up to 4 h). ACTH was bioassayed using isolated rat adrenocortical cells; ACTH-(1-24) served as the standard for expressing the data. Results with 16-h preincubated cells were as follows: CRF induced ACTH secretion in a concentration-dependent manner: ED50 and maximal stimulatory concentrations were 1.0 nM and 5.0 nM, respectively. CRF (10 nM) induced significant ACTH secretion within 10 min of incubation; maximal secretion (370% over basal value) was attained at 2 h. Dexamethasone (DEX) inhibited basal and CRF-induced ACTH secretion in a concentration-dependent manner; half-maximal inhibitory and maximal inhibitory concentrations were approximately 10 nM and 1 microM, respectively. In addition, DEX (10 microM) acutely (within 2 h) inhibited maximal CRF-induced ACTH secretion by 46%. 8-Bromo-cAMP (1 mM) also induced ACTH secretion, and DEX inhibited this secretion with a potency equivalent to that for CRF-induced ACTH secretion. In contrast to the effect of CRF, high concentrations (100 nM) of ovine LHRH, TRH, and synthetic human pancreatic GH-releasing factor (1-32) failed to induce significant ACTH secretion, thus suggesting that the effect of CRF was peptide specific. Domestic fowl pituitary cells cultured for 48 h before treatment also responded to CRF but not to any greater extent than that of 16-h preincubated cells. In contrast to 16-h preincubated cells or 48-h cultured cells, 2-h preincubated cells had high basal values of ACTH secretion that may have partially diminished or masked the actions of CRF. These data suggest that 1) CRF is a potent and specific stimulator of ACTH secretion by domestic fowl pituitary cells and 2) 16-h preincubated cells or 48-h cultured cells are amenable for other in vitro investigations on the regulation of avian ACTH secretion.