Off-resonance saturation caused a reduction of the 3.04 ppm NMR signal from the methyl protons of creatine in rat hindleg skeletal muscle. 1 H-NMR spectra were recorded over a 200 kHz range of off-resonance saturation frequencies. The span of frequencies over which the creatine signal was reduced greatly exceeded that expected for direct saturation by the offresonance RF-field. This suggests that there is a motionally restricted proton pool which exchanges magnetization with the free creatine pool. The experimental data were fitted to characterize the immobilized proton pool and the exchange kinetics, using a two-pool exchange model. The immobile pool was estimated to amount to ca. 2.5% of the mobile pool of free creatine, while the rate of exchange between the mobile and immobile configurations is ca. 2.3 sec -1 . After depletion of phosphocreatine by termination of the animal, the MT effect on the creatine methyl protons remained unchanged. This indicates that phosphocreatine and creatine both contribute to the MT phenomenon. Selective saturation of the mobile water pool also led to a reduction in the intensity of the total creatine methyl signal, suggesting that water and creatine are magnetically coupled via a macromolecular interface. The precise mechanism responsible for and the biological significance of the pronounced creatine magnetization transfer effect in rat skeletal muscle remains to be established. Magn Reson Med 42:665