Gene therapy utilizing leukocytes is an unexplored therapeutic strategy for targeting tissue-type plasminogen activator (t-PA) to fibrin and sites of inflammation. In this study, five cationic lipids were observed to enhance the adenovirus (Ad)-mediated expression of t-PA in human peripheral blood mononuclear cells (PBMCs) in a dose-dependent manner between 1000 and 15,000 lipid molecules per Ad particle (efficiency:LipofectAMINE > GenePORTER > Effectene > SuperFect > DMRIE-C). PBMCs treated with Ad/t-PA * LipofectAMINE complexes displayed elevated t-PA expression over a 4-day period and the t-PA-expressing cells facilitated the lysis of plasma clots in vitro. Functional and immunologic assays revealed that the Ad * LipofectAMINE infection protocol did not affect monocyte adhesion in vitro or elevate the expression of procoagulant activity, interleukin 8, or tumor necrosis factor alpha. The potential of this system was documented with an in vivo rat model system that involved the injection of lipopolysaccharide into the peritoneal cavity to induce an inflammatory response. Infusion of Ad/t-PA-infected rat PBMCs into the vasculature of lipopolysaccharide-treated animals was found to increase local fibrinolytic activity by 4-fold. These data provide a framework for utilizing adenovirus to transfer genes into PBMCs.