Increased expression of plasminogen activator inhibitor -1 (PAI-1) in adipose tissues is thought to contribute to both the cardiovascular and metabolic complications associated with obesity. Tumor necrosis factor alpha (TNF-alpha) is chronically elevated in adipose tissues of obese rodents and humans and has been directly implicated to induce PAI-1 in adipocytes. In this study, we used 3T3-L1 adipocytes to examine the mechanism by which TNF-alpha up-regulates PAI-1 in the adipocyte. Acute (3 h) and chronic (24 h) exposure of 3T3-L1 adipocytes to TNF-alpha induces PAI-1 mRNA by increasing the rate of transcription of the PAI-1 gene, and de novo protein synthesis is not required for this process. Although the p44/42 and PKC signaling pathways appear to be significant in the induction of PAI-1 mRNA in response to acute treatment with TNF-alpha, the more dramatic induction of PAI-1 mRNA observed in response to chronic exposure of adipocytes to TNF-alpha was mediated by these and additional signaling molecules, including p38, PI3-kinase, tyrosine kinases, and the transcription factor NF-kappaB. Moreover, the dramatic increase in PAI-1 observed after chronic exposure of adipocytes to TNF-alpha was accompanied by increased metabolic insulin resistance. Finally, we demonstrate that the PKC pathway is also central for PAI-1 induction in response to insulin and transforming growth factor-beta (TGF-beta), two additional molecules which are elevated in obesity and shown to directly induce PAI-1 in the adipocyte. The understanding of the mechanism of regulating PAI-1 expression in the adipocytes at the molecular level provides new insight to help identify novel targets in fighting the pathological complications of obesity.