Skeletal muscle fiber heterogeneity among muscle groups is well known; however, laterality of muscle metabolism has not been addressed. In the present studies, metabolite concentrations in left and right gastrocnemius, tibialis anterior, quadriceps, and soleus muscles and their response to exogenous insulin have been compared in fasted awake rats. The results indicated that the concentrations of muscle free glycerol (P >.4), glycerol 3-phosphate (P >.1) nonesterified fatty acids (NEFA) (P >.6) and intramyocellular triglycerides (imcTG) (P >.08) are comparable between left and right of the same muscle, and are similar among mixed glycolytic-oxidative muscles. The concentration of free glycerol in soleus responded to exogenous insulin in a pattern distinct to that seen for the mixed muscles. The results support interchangeable use of left and right side of same muscles, and probably among different muscles of similar fiber type, but not muscles of different fiber types.