OBJECTIVE Engineered overexpression of tissue plasminogen activator (tPA) in vascular cells has been proposed as a means to decrease intravascular thrombosis; however, tPA gene transfer has augmented intimal hyperplasia in vivo in some studies. The purpose of this study was to define in vitro the effect of tPA gene transfer on smooth muscle cells (SMCs). METHODS Human SMCs were retrovirally transduced with the tPA gene (SMCs/tPA). RESULTS In the absence of plasminogen, no statistical differences in proliferation, migration, and morphology were observed between SMCs/tPA and SMCs. In the presence of plasminogen, many differences became apparent. Matrix metalloproteinase-2 (MMP-2) activation was 10-fold higher in SMCs/tPA than in SMCs. This activation was inhibited by aprotinin, a plasmin inhibitor. Collagen degradation increased sevenfold in SMCs/tPA. SMCs/tPA contracted dramatically in the presence of plasminogen. This cell contraction, indicative of extracellular matrix degradation, was blocked by aprotinin and partially inhibited by MMP inhibitors. SMC/tPA-conditioned medium induced significantly more SMC proliferation. The migration of SMCs/tPA through a porous membrane significantly exceeded untransduced SMCs. CONCLUSIONS Over-expression of tPA in SMCs results in increased extracellular matrix degradation and can promote cell proliferation and migration. This effect is mediated via plasmin, which further activates MMP-2. CLINICAL RELEVANCE TPA has been clinically used as a thrombolytic agent in the treatment of acute thrombotic disorders. Transferring the tPA gene into vascular cells as a strategy of gene therapy has been proposed to enhance fibrinolytic capability and therefore inhibit thrombosis and restenosis after vascular interventions. The mechanism(s) by which tPA affects SMC proliferation and vascular remodeling has not been thoroughly characterized. This study unveils the relationship between thrombolytic activity and intimal hyperplasia by showing how the elevated expression of tPA affects the vascular remodeling. This study underscores that the overexpression of an enzyme thought beneficial to blood flow can potentially compromise blood flow by altering the biology of the cell engineered to express it. The results are important to the rational engineering of bioactive grafts with better patency. A new strategy to enhance the thrombolytic ability of a vascular surface without inducing excessive neointimal hyperplasia is proposed.