NO plays a role in a variety of in vitro models of angiogenesis, although confounding effects of NO on non-endothelial tissues make its role during in vivo angiogenesis unclear. We therefore examined the effects of NO on two physiological models of angiogenesis in mouse skeletal muscle: (1) administration of prazosin (50 mg l(-1)) thereby increasing blood flow; and (2) muscle overload from surgical ablation of a functional synergist. These models induce angiogenesis via longitudinal splitting and capillary sprouting, respectively. Administration of N-G-nitro-L-arginine (L-NNA) abolished the increase in capillary to fibre ratio (C:F) in response to prazosin administration, along with the increases in luminal filopodia and large endothelial vacuoles. L-NNA prevented luminal filopodia and vacuolisation in response to extirpation, but had no effect on abluminal sprouting, and little effect on C:F. Comparison of mice lacking endothelial (eNOS(-/-)) and neuronal NO synthase (nNOS(-/-)) showed that longitudinal splitting is eNOS-dependent, and Western blotting demonstrated an increase in eNOS but not inducible NOS (iNOS) expression. These data show that there are two pathways of physiological angiogenesis in skeletal muscle characterised by longitudinal splitting and capillary sprouting, respectively. NO generated by eNOS plays an essential role in splitting but not in sprouting angiogenesis, which has important implications for angiogenic therapies that target NO.