—One mechanism by which high density lipoproteins (HDLs) exert their protective effect against coronary artery disease could be related to the induction of prostacyclin (PGI₂) release in the vessel wall. We have recently shown that HDL increases PGI₂ production in rabbit smooth muscle cells (RSMCs) and that this increase is dependent on cyclooxygenase-2 (Cox-2). Here we analyze the mechanism by which rabbit HDL induces PGI₂ release in RSMCs. Our results show that although HDL₂ and HDL₃ share a similar capacity to induce Cox-2 protein levels, HDL₃ stimulates a higher PGI₂ release than does HDL₂, probably because of their relative arachidonate contents. Acetylsalicylic acid pretreatment (300 μmol/L, 30 minutes) significantly reduced the HDL-induced PGI₂ release, suggesting that both preexisting and induced Cox-2 activities were involved in the HDL effect. Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Cox-1 protein levels were not altered by HDL. Dexamethasone (2 μmol/L), which also inhibited the HDL-induced PGI₂ release, reduced significantly both Cox-2 mRNA and protein levels without affecting cPLA₂ and Cox-1 protein levels. In addition, methylarachidonyl fluorophosphonate, a potent inhibitor of cPLA₂, did not produce any effect on HDL-induced PGI₂ release. In the presence of cycloheximide, Cox-2 mRNA levels were induced by HDL and inhibited by dexamethasone, suggesting that HDL and dexamethasone work in the absence of de novo protein synthesis. These results indicate an early effect of HDL on PGI₂ biosynthesis, specifically increasing Cox-2. PD98059, an inhibitor of mitogen-activated protein kinase kinase, completely inhibited HDL-induced PGI₂ release, whereas GF109203X, a protein kinase C inhibitor, had no effect. Thus, HDL induces PGI₂ synthesis by a mechanism dependent on the mitogen-activated protein kinase pathway but independent of protein kinase C.