Stimuli of prostacyclin (PGI2) biosynthesis such as thrombin, bradykinin, histamine, and A23187 increase guanosine 3',5'-cyclic monophosphate (cyclic GMP) levels in primary monolayer cultures of human umbilical vein endothelium by about twofold. This effect is dependent on the presence of extracellular Ca2+. Increases of about tenfold are observed when cyclic GMP phosphodiesterase activity is inhibited, which suggests that the observed increases in cyclic GMP involve the activation of guanylate cyclase. Activation of guanylate cyclase appears to involve an early event in the induction of PGI2 biosynthesis, as neither arachidonic acid nor its metabolites stimulate cyclic GMP accumulation. Although activators of guanylate cyclase such as atriopeptin III, sodium nitroprusside, and tert-butylhydroperoxide increase cyclic GMP levels by approximately 2-3-fold, they do not stimulate or modulate PGI2 production. We conclude that cyclic GMP does not play a primary role in mediating the induction or regulation of PGI2 biosynthesis in vascular endothelium.