The utility of adenoviral vectors, currently used in cardiovascular gene transfer protocols, is limited by the brevity of transgene expression and by antiadenoviral immune responses. The effect of preexisting antiadenoviral immunity on intracardiac gene transfer or its modulation by nitric oxide is unknown. Adenoviral vectors, expressing the firefly luciferase gene (AdLuc) or the human nitric oxide synthase 3 (NOS3) gene (AdNOS3), were infused into the great cardiac vein of naive pigs or immunized pigs. Pigs were immunized by intravenous injection of control virus AdRR5 and the resulting neutralizing antibody titers (median, 1:178; p < 0.0001 vs. baseline) were similar to preexisting titers in 54% of randomly selected coronary artery bypass graft patients. In naive animals distribution of transgene expression in the left ventricular free wall was focal. In immunized pigs myocardial luciferase expression 3 days after AdLuc gene transfer was more than 1000-fold lower than in naive pigs, whereas no change in NOS3 transcript levels was detected after AdNOS3 gene transfer. Severe, grade III-IV mononuclear cell infiltration and myocyte apoptosis were observed in four of five AdLuc-infected, immunized animals, compared with low-level inflammation and apoptosis in five of six AdNOS3-infected pigs. Coinfusion of AdLuc and AdNOS3 in immunized pigs resulted in spatially colocalized transgene expression, reduced T cell-mediated inflammation, and myocyte apoptosis and was associated with 200-fold greater median reporter transgene expression levels in the subendocardium (1.0 x 10(3) light units [LU]/mg protein, n = 8, vs. 4.5 x 10(1) LU/mg protein in AdLuc- and AdRR5-coinfected pigs, n = 7, p = 0.02). Preexisting antiadenoviral immunity abrogates myocardial gene expression in pigs and is associated with severe inflammation and myocyte apoptosis. Intracardiac NOS3 gene transfer may reduce these barriers to adenovirus-mediated myocardial gene transfer.