Exercise is dependent on adequate oxygen supply for mitochondrial respiration in both cardiac and locomotor muscle. To determine whether skeletal myofiber VEGF is critical for regulating exercise capacity, independent of VEGF function in the heart, ablation of the VEGF gene was targeted to skeletal myofibers (skmVEGF-/-) during embryogenesis (~E9.5), leaving intact VEGF expression by all other cells in muscle. In adult mice, VEGF levels were decreased in the soleus (by 65%), plantaris (94%), gastrocnemius (74%), EDL (99%) and diaphragm (64%) (P<0.0001, each muscle). VEGF levels were unchanged in the heart. Treadmill speed (WT 86±4cm/sec, skmVEGF-/- 70±5cm/sec, P=0.006) and endurance (WT 78±24min, skmVEGF-/- 18±4min, P=0.0004) were severely limited in skmVEGF-/- mice in contrast to minor effect of conditional skmVEGF gene deletion in the adult. Body weight was also reduced (WT 22.8±1.6g, skmVEGF-/-, 21.1±1.5, P=0.02), but the muscle mass/body weight ratio was unchanged. The capillary/fiber ratio was lower in skmVEGF-/- plantaris (WT 1.51±0.12, skmVEGF-/- 1.16±0.20, P=0.01), gastrocnemius (WT 1.61±0.08, skmVEGF-/- 1.39±0.08, P=0.01), EDL (WT 1.36±0.07, skmVEGF-/- 1.14±0.13, P=0.03) and diaphragm (WT 1.39± 0.18, skmVEGF-/- 0.79±0.16, P=0.0001) but, not in soleus. Cardiac function (heart rate, maximal pressure, maximal dP/dt, minimal dP/dt,) in response to dobutamine was not impaired in anesthetized skmVEGF-/- mice. Isolated soleus and EDL fatigue times were 16% and 20% (P<0.02) longer, respectively, in skmVEGF-/- mice than the WT group. These data suggest that skeletal myofiber VEGF expressed during development is necessary to establish capillary networks that allow maximal exercise capacity.