Histamine stimulates the production of prostacyclin (PGI2) in cultured human endothelial cells. We have examined the cell specificity of histamine-mediated PGI2 synthesis in primary and subcultured human cells. Venous and arterial smooth muscle cells and skin fibroblasts synthesized PGI2 from exogenous arachidonic acid, but they did not synthesize a significant amount of PGI2 when treated with histamine. Endothelial cells, however, produced similar amounts of PGI2 in response to histamine and arachidonic acid. Thrombin also stimulates PGI2 production in endothelial cells. Histamine and thrombin yielded an additive production of PGI2 when added simultaneously to endothelial cells. When histamine and thrombin were added sequentially, the amount of PGI2 produced was not additive but equaled the amount characteristic of the first agonist alone. Following an initial treatment with histamine, endothelial cells were unable to respond to histamine for 3 hr, after which the PGI2 biosynthetic response rapidly returned to normal by 4 1/2 hr. When the initial histamine treatment was carried out under mildly alkaline conditions, the complete return of activity was delayed to 8 hr after treatment. The synthesis of PGI2 from exogenous arachidonic acid was unaffected by prior treatment with histamine. Recovery of histamine-mediated PGI2 production was not dependent on protein synthesis but required a component of fetal calf serum that is nondialyzable and moderately heat stable. Thus endothelial cell PGI2 synthesis in responses to a physiologic agonist is subject to several levels of regulation, reflecting not only intracellular events but also the extracellular environment.