Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of α-syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates α-adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle–derived NO. To test this hypothesis, we performed experiments in α-syntrophin null mice and in transgenic mice expressing a mutated α-syntrophin lacking the PDZ domain (ΔPDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of α-adrenergic vasoconstriction was greatly impaired in the contracting muscles of the α-syntrophin null mice and transgenic ΔPDZ mice compared with wild-type mice and transgenic mice expressing full-length α-syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein α-syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.