SRIF inhibits ACTH secretion by AtT20/D16v (D16) mouse pituitary cells stimulated by high (50 mM) extracellular concentrations of K+ or by divalent cation ionophores. Although stimulation of ACTH secretion by K+ requires extracellular Ca+2, the response is invariant over medium Ca+2 concentrations of 0.003-1 mM; with Ca+2 concentrations from 1-5 mM there is a dramatic amplification of the secretory response. SRIF at concentrations of 10(-8) M completely inhibits the secretory response to K+ at Ca+2 concentrations between 0.2 and 1 mM; with increasing medium Ca+2 above 1 mM there is a progressive attenuation of SRIF-inhibition. At concentrations of 5 mM, Ca+2 alone can serve as an ACTH secretagogue. The ionophore ionomycin stimulates ACTH secretion in a Ca+2-dependent manner with a half-maximal effect at 5 X 10(-6) M ionomycin. The secretory response to ionomycin and to X537A is inhibited by at least 50% by SRIF. The secretory response to K+ is accompanied by a rapid and sustained increase in 45Ca+2 uptake, whereas the ionophores ionomycin, X537A, and A23187 increase Ca+2 efflux. SRIF does not affect Ca+2 movement across D16 cell membranes in response to either K+ or ionophores. These results show that an increase in intracellular Ca+2 is an effective stimulus to ACTH secretion by D16 cells and inhibition of ACTH secretion by SRIF is not effected by interference with the stimulus-elicited increase in intracellular Ca+2.