Stimulation of steroid production by isolated cat adrenocortical cells is partially dependent upon the presence of extracellular Ca 2+ when elicited by prostacyclin (PGI 2) and completely dependent upon extracellular Ca 2+ when elicited by corticotropin. TMB-8, an intracellular Ca 2+ antagonist, completely blocked PGI 2-evoked steroid output in the absence of external Ca 2+; this inhibition was partially reversed by the addition of Ca 2+. The increase in secretion caused by corticotropin or PGI 2 in the presence of Ca 2+ was also reduced in a dose-dependent manner by TMB-8. The steroidogenic action of pregnenolone, which is induced by a Ca 2+ independent mechanism, was not blocked by TMB-8, either in the presence or absence of Ca 2+. Corticotropin significantly potentiated the Ca 2+-independent aspect of PGI 2 action. These studies provide evidence for an internal, PGI 2-sensitive Ca 2+ store in cat adrenocortical cells.