Phase-modulated rotating-frame imaging, a localization technique for phosphorus nuclear magnetic resonance spectroscopy, has been applied to obtain information on heterogeneity of phosphorus-containing metabolites in skeletal muscle of the rat in vivo. The distal muscles of the rat hindlimb have been studied at rest and during steady-state isometric twitch contraction; the use of a transmitter surface coil and an electrically isolated, orthogonal receiver Helmholtz coil ensure accurate spatial assignment (1 mm resolution). At rest, intracellular pH was higher and PCr/(PCr + Pi) was lower in deeper muscles compared with superficial muscle of the distal hindlimb. Upon steady-state stimulation, the relatively more alkaline pH of deep muscle was maintained, whereas greater changes in PCr/(PCr + Pi) and Pi/ATP occurred in the superficial muscle layer. This method allows rapid (75 min for each spectral image) acquisition of quantitative information on metabolic heterogeneity in vivo.