Rat extensor digitorum longus muscles were overloaded by stretch after removal of the synergist tibialis anterior muscle to determine the relationship between capillary growth, muscle blood flow, and presence of growth factors. After 2 wk, sarcomere length increased from 2.4 to 2.9 μm. Capillary-to-fiber ratio, estimated from alkaline phosphatase-stained frozen sections, was increased by 33% (P < 0.0001) and 60% (P < 0.01), compared with control muscles (1.44 ± 0.06) after 2 and 8 wk, respectively. At 2 wk, the increased capillary-to-fiber ratio was not associated with any changes in mRNA for basic fibroblast growth factor (FGF-2) or its protein distribution. FGF-2 immunoreactivity was present in nerves and large blood vessels but was negative in capillaries, whereas the activity of low-molecular endothelial-cell-stimulating angiogenic factor (ESAF) was 50% higher in stretched muscles. Muscle blood flows measured by radiolabeled microspheres during contractions were not significantly different after 2 or 8 wk (132 ± 37 and 177 ± 22 ml ⋅ min⁻¹ ⋅ 100 g⁻¹, respectively) from weight-matched controls (156 ± 12 and 150 ± 10 ml ⋅ min⁻¹ ⋅ 100 g⁻¹, respectively). Resistance to fatigue during 5-min isometric contractions (final/peak tension × 100) was similar in 2-wk overloaded and contralateral muscles (85 vs. 80%) and enhanced after 8 wk to 92%, compared with 77% in contralateral muscles and 67% in controls. We conclude that increased blood flow cannot be responsible for initiating expansion of the capillary bed, nor does it explain the reduced fatigue within overloaded muscles. However, stretch can present a mechanical stimulus to capillary growth, acting either directly on the capillary abluminal surface or by upregulating ESAF, but not FGF-2, in the extracellular matrix.