Human normal plasma stimulates prostacyclin (PGI2) production by vascular cells. This plasma activity may be greatly modified in different pathological conditions. The purpose of this study was to characterize some aspects of the mechanism of action of plasma in modulating PGI2 release. Cultured rat aortic smooth muscle cells were used. Citrated plasma from healthy donors stimulated PGI2 production in a concentration-dependent way. Plasma-derived serum containing increasing concentrations of platelets had the same PGI2 stimulating activity as citrated plasma. Plasma stimulation of PGI2 production was accompanied by release of endogenously incorporated arachidonic acid (AA) from the cell membrane. Similarly to AA, plasma induced PGI2 synthesis only once, a second or third challenge producing a reduced response from the cells. Cells stimulated twice with plasma responded to AA like unstimulated cells while cells stimulated twice with AA were poorly responsive to subsequent stimulation with plasma. When the cells were repeatedly stimulated with AA in the presence of plasma no refractoriness was apparent. This study suggests that plasma increases PGI2 synthesis by the release of endogenous substrate from the cell membrane and by protecting the cells from self-inactivation during AA conversion to prostaglandins.