The observation of a previously unidentified peak in localized 1H magnetic resonance (MR) spectra of human muscle during and after a work load is reported. Basic NMR properties of this resonance, as well as physiologic circumstances of its observation, suggest that it is due to the acetyl group of acetylcarnitine. The relatively large pool of muscular carnitine acts as a buffering system stabilizing the ratio of acetylated to free coenzyme A. Free carnitine can be acetylated to a large extent whenever a mismatch occurs between the fluxes through pyruvate dehydrogenase and the TCA cycle. Results of initial applications of 1H MR spectroscopy in several muscles and under different exercise regimens are in agreement with earlier invasive measurements of acetylcarnitine. It is demonstrated that the detailed dynamics of acetyl group formation are now likely to be observable non-invasively in humans by localized 1H magnetic resonance spectroscopy on standard MR imaging systems, and that acetylcarnitine buffering as a function of exercise type, oxygenation states, diet and pathology could thus be studied repeatedly and in various muscle groups with much improved temporal resolution.