Capillary-to-fiber perimeter ratio is a morphometric estimate of muscle capillarity in transverse sections which accounts for the three-dimensional arrangement of the capillary network. We compared different methods for estimating capillary-to-fiber perimeter ratio in muscles with large differences in fiber size and capillary density or geometry (hummingbird and bat flight muscle, bat hindlimb, tuna red muscle and rat M. soleus). There was no significant difference between light and electron microscopy estimates of capillary-to-fiber perimeter ratio by direct intersection-counting in transverse sections. Calculated values via capillary surface per fiber volume and fiber cross-sectional area/perimeter were not significantly different from those obtained by direct intersection-counting in muscle transverse sections in any muscle. A closer estimate of capillary-to-fiber perimeter ratio to that obtained by direct intersection-counting in transverse sections was calculated via capillary surface density than capillary length per fiber volume and capillary diameter, possibly because of the greater number of variables used to calculate capillary-to-fiber perimeter ratio via capillary length density and diameter. A greater capillary-to-fiber perimeter ratio was found in hummingbird and bat flight muscle than in the other muscles, consistent with an important role of the capillary-to-fiber interface in determining O2 flux rates and measurements of mitochondrial respiratory rates in flying hummingbird that are about two times greater than those in locomotry muscles of mammals running at VO2max.