This study quantifies myocardial transfection following percutaneous retrograde coronary venous delivery (RCVD) of a plasmid encoding human Del‐1. RCVD of Del‐1, GFP plasmid, or marker dye was conducted in 14 pigs. After selective cannulation of a coronary vein, a delivery site was confirmed by contrast injection and myocardial blush. Ten milliliters of plasmid hDel‐1 or GFP was administered. Animals were euthanized 3 and 7 days post‐RCVD. hDel‐1 gene expression was evaluated by quantitative RT‐PCR. An average myocardial expression of 4.5 × 10⁵ copies hDel‐1/μg total RNA was observed within the approximately 5 × 5 cm² target tissue of the left ventricle. GFP expression was detected by fluorescent microscopy. hDel‐1 protein expression was confirmed by immunohistochemistry. Regionalized myocardial expression was found in all pigs. hDel‐1 RNA was not found in distant tissues except in the three pigs with prominent venovenous washout (PVW). These levels were 3 to 4 log unites lower than those found in myocardium. Single retrograde coronary venous administration resulted in efficient regional myocyte transfection of hDel‐1 and GFP. This method may be useful and clinically feasible for myocardial angiogenesis therapy. Cathet Cardiovasc Intervent 2003;58:207–211. © 2003 Wiley‐Liss, Inc.