Plasminogen activator inhibitor type-1 (PAI-1) regulates fibrinolytic activity and mediates vascular atherothrombotic disease. Endothelial cells (ECs) synthesize and secrete PAI-1, but the intracellular signaling pathways that regulate PAI-1 expression are not entirely known. We hypothesize that the phosphatidylinositol 3-kinase (PI3K)/protein kinase Akt pathway, which regulates endothelial function, could modulate PAI-1 expression in ECs. Cultured bovine aortic and human saphenous vein ECs were stimulated with tumor necrosis factor (TNF)-, angiotensin II, insulin, or serum, and PAI-1 expression was determined by Northern and Western analyses. Inhibition of PI3K with either wortmannin or LY294002 enhanced PAI-1 expression induced by these extracellular stimuli. Similarly overexpression of a dominant-negative mutant of PI3K or Akt increased TNF- and insulin-induced PAI-1 expression. The increase in PAI-1 was due to transcriptional and post-transcriptional mechanisms as PI3K inhibitors increased PAI-1 promoter activity and mRNA stability. The induction of PAI-1 by TNF and insulin is mediated, in part, by extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK). PI3K inhibitors augmented TNF- and insulin-induced phosphorylation of these MAPKs. Simvastatin, a HMG-CoA reductase inhibitor which is known to activate PI3K/Akt, block TNF- and insulin-induced PAI-1 expression. Treatment with PI3K inhibitors reversed the inhibitor effects of simvastatin on TNF- and insulin-induced PAI-1 expression. These findings indicate that PI3K/Akt pathway acts as a negative regulator of PAI-1 expression in ECs, in part, through the downregulation of MAPK pathways. These results suggest that factors, which activate the PI3K/Akt pathway in EC, may have therapeutic benefits for atherothrombotic vascular disease.