Increased expression of plasminogen activator inhibitor type 1 (PAI-1) is associated with decreased apoptosis of neoplastic cells. We sought to determine whether PAI-1 alters apoptosis in vascular smooth muscle cells (VSMC) and, if so, by what mechanisms. A twofold increase in the expression of PAI-1 was induced in VSMC from transgenic mice with the use of the SM-22α gene promoter (SM22-PAI+). Cultured VSMC from SM22-PAI+ mice were more resistant to apoptosis induced by tumor necrosis factor plus phorbol myristate acetate or palmitic acid compared with VSMC from negative control littermates. Both wild type (WT) and a stable active mutant form of PAI-1 (Active) inhibited caspase-3 amidolytic activity in cell lysates while a serpin-defective mutant (Mut) PAI-1 did not. Similarly, both WT and Active PAI-1 decreased amidolytic activity of purified caspase-3, whereas Mut PAI-1 did not. WT but not Mut PAI-1 decreased the cleavage of poly-[ADP-ribose]-polymerase (PARP), the physiological substrate of caspase-3. Noncovalent physical interaction between caspase-3 and PAI-1 was demonstrable with the use of both qualitative and quantitative in vitro binding assays. High affinity binding was eliminated by mutations that block PAI-1 serpin activity. Accordingly, attenuated apoptosis resulting from elevated expression of PAI-1 by VSMC may be attributable, at least in part, to reversible inhibition of caspase-3 by active PAI-1. © 2004 Wiley-Liss, Inc.