The difference in concentration of phosphorylated metabolites in muscles with different fiber composition was studied in vivo by localized 31P nuclear magnetic resonance spectroscopy in the rat hindlimb 120-160 microliters volumes were selected in regions containing the soleus and gastrocnemius muscles. Concentrations of phosphocreatine (PCr), adenosine triphosphate and inorganic phosphate (Pi) were determined and intracellular pH was calculated in the respective muscle groups. The highest level of PCr was found in the gastrocnemius muscle, containing 30.7 mmoles/dm3 tissue compared to 22.3 mmoles/dm3 in the soleus muscle. Pi was significantly lower in gastrocnemius (1.9 mmoles/dm3) than in soleus (3.2 mmoles/dm3). The ATP concentration was 6.7 and 6.4 mmoles/dm3 and pH was determined to 7.11 and 7.09 in the gastrocnemius and soleus muscle, respectively. Our NMR data show that it is possible to measure high-energy phosphates with precision in small localized volumes with the ISIS method using a Helmholtz coil. Earlier biochemical data are confirmed by these in vivo NMR results. Localized in vivo 31P NMR spectroscopy can contribute to the understanding of the underlying mechanisms of several metabolic events in different regions of the tissue. The method can be used for future studies of varying ischemia tolerance, varying degrees of adaptation to exercise with regard to oxidative capacity, and pH compartmentation in muscles with different fiber composition.