We have used two-dimensional gel electrophoresis (2-DE) and mass spectrometry (MS) to study the expression of contractile and regulatory proteins in human vastus lateralis and deltoideus muscles, in order to understand protein turnover and isoform switching in muscles with the same fiber-type composition but different functional properties. We demonstrate a two- to six-fold overexpression of enzymes associated with glycolysis, the tricarboxylic acid cycle, oxidative phosphorylation, and substrate transport in vastus lateralis compared to deltoideus. Expression levels of contractile protein isoforms correlated to the proportion of slow-twitch fibers in deltoideus compared to vastus lateralis are consistent with the different contractile properties of the two muscles. Two proteins involved in free radical homeostasis were differentially expressed, suggesting a direct relationship between radical scavenging and the muscle function. The application of 2-DE and MS to studies of muscle physiology thus offers a more comprehensive assessment of the molecular determinants of muscle function than traditional approaches.