Previously, we demonstrated that conditioned medium (CM) from cultures of human diploid fibroblast cells contains a factor that stimulates the production of prostacyclin (PGI2) by cultured bovine aortic endothelial cells (BAEC). To study the mechanism by which CM stimulates PGI2 production, we measured the effect of removal of extracellular calcium (Ca2+) on the concentration of cytosolic Ca2+ and on the production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), a stable metabolite of PGI2. The CM-induced production of 6-keto-PGF1 alpha was dependent on extracellular Ca2+ and did not require nascent protein synthesis. Application of CM to BAEC induced a transient increase in cytosolic Ca2+ concentration that was dependent on extracellular Ca2+. Bradykinin induced the production of 6-keto-PGF1 alpha by BAEC. However, bradykinin induced an increase in cytosolic Ca2+ concentration in the presence or absence of extracellular Ca2+. Voltage dependent Ca2+ channel blocker (verapamil, diltiazem) did not inhibit either the CM-induced increase in cytosolic Ca2+ or the production of 6-keto-PGF1 alpha by BAEC. These data suggest that CM increases the cytosolic Ca2+ concentration and stimulates PGI2 production by BAEC. The increase in cytosolic Ca2+ concentration occurred via the influx of extracellular Ca2+ independent of L-type Ca2+ channels blocked by verapamil or diltiazem.