Gene therapy strategies for the prevention of restenosis postangioplasty are promising. Nonviral gene transfer to the arterial wall in vivo has so far been limited by poor efficiency. This study aimed to optimize transfection of primary vascular smooth muscle cells using cationic nonviral formulations based on cholesterol derivates (DC-, DAC-, DCQ-, and Sp-Chol), double-chained amphiphils (LipofectAMINE, DOTMA, DOSGA, DOSPER, and DOCSPER), or heterogeneous reagents (Superfect, Effectene, and Tfx-50). Estimation of transfection efficiencies was performed using galactosidase assays at different ratios of transfection reagent to plasmid DNA with reporter gene. Toxicity was monitored by analyzing cell metabolism. Transfer efficiency and safety were determined in a porcine restenosis model for local gene therapy using morphometry, histology, galactosidase assays, and reverse-transcriptase polymerase chain reaction. The highest in vitro transfection efficiency was achieved using the recently developed DOCSPER liposomes, with transfer rates of at least 20% in vascular smooth muscle cells. Transfer efficiency was further enhanced up to 20% by complexing with poly-L-lysine. Transfection efficiency in vivo in a porcine restenosis model was up to 15% of adventitial cells using DOCSPER versus 0.1% using LipofectAMINE. Toxicity in vivo and in vitro was lowest using DOCSPER. Increased biological effects were demonstrated following optimization of transfer conditions.