We have shown that c-Src plays a role in shear stress stimulation of endothelial nitric oxide synthase (eNOS) expression in cultured cells. To examine the role of c-Src in vivo, we exercised C57Blk/6 and c-Src heterozygous (c-Src(+/-)) mice on a treadmill for 3 wk. Western analysis demonstrated that c-Src(+/-) mice express less than one-half the normal amount of c-Src. Exercise increased heart rate and blood pressure to identical levels in both strains as determined using radiotelemetry. Exercise training increased eNOS protein >2-fold in the aorta and 1.7-fold in the heart in C57Blk/6 mice but had no effect on eNOS protein levels in c-Src(+/-) mice. In contrast to exercise, treatment of mice with mevastatin, which stimulates expression of eNOS posttranscriptionally, increased eNOS protein in both strains. Training also increased aortic extracellular superoxide dismutase protein expression, which is regulated by nitric oxide, in C57Blk/6 mice but not in c-Src(+/-)mice. These data indicate that c-Src has an important role in modulating vascular adaptations to exercise training, in particular increasing eNOS and extracellular superoxide dismutase protein expression.