In light of previous studies which have implicated prostaglandin (PG) formation as a link in ACTH-induced steroid production by isolated cat adrenocortical cells, experiments were carried out to provide additional information regarding the role of PGs in adrenal steroidogenesis and their interactions with calcium and cyclic AMP. Perfusion of cat adrenal glands with Locke's solution plus beta(1-24)-ACTH resulted in an immediate increase in PGF2alpha release, which rapidly declined to basal levels after the stimulus was withdrawn. In contrast, maximal rates of steroid release were manifest some 30 min after removal of ACTH. ACTH and its onitrophenyl sulfenyl derivative (NPS-ACTH) increased PG (PGF2alpha and PGE2) and steroid release by trypsin-dispersed cat cortical cells, but NPS-ACTH, unlike ACTH, did not augment cortical cyclic AMP levels. In this same preparation, indomethacin completely blocked ACTH and NPS-ACTH facilitated PGF2alpha and PGE2 release but failed to suppress steroid release markedly. Calcium-deprivation blocked PG and steroid release evoked by these two polypeptides, and depressed PG release elicited by monobutyryl cyclic AMP (bcAMP) without affecting steroid release. These experiments offer additional evidence to support the concept that PGs play a role in the mode of action of ACTH; however, they do not appear to be obligatory intermediates in the steroidogenic process. The importance of calcium in regulating PG formation is discussed with special regard for the idea that this cation has a direct action on the enzyme systems which control PG synthesis.