Using in vivo proton magnetic resonance spectroscopy (1H-MRS), a new peak resonating at 2.13 ppm post-exercise has been attributed in the literature to the acetyl groups of acetylcarnitine. Since this peak is inconsistently generated by various submaximal exercise regimens, this study aimed at (a) verification of the previous chemical assignment, (b) determination of exercise conditions necessary for its induction, and (c) documentation of the recovery kinetics through 60 minutes following exercise. Ten healthy males (31 +/- 4 yr) cycled continuously for 45 minutes with intensity alternating between 50% (3 min) and 110% (2 min) of ventilatory threshold (VT). 1H-MR spectra were acquired from the vastus lateralis before and for 60 minutes following exercise. The peak at 2.13 ppm was not quantifiable at rest in any subject. However, it was present in all subjects following intense exercise (p < 0.0001), and expressed the chemical characteristics of an acetyl-containing compound. The estimated concentration, accumulation with high-intensity exercise, the presence as a single peak at 2.13 ppm, and the chemical shift were all consistent with the chemical and biophysical characteristics of acetyl groups associated with acetylcarnitine. This study provides further evidence that acetyl groups are robustly generated by intense exercise, and that the accumulation of acetyl groups in healthy subjects is dependent on the degree of exercise intensity. 1H-MRS may be used for the noninvasive study of muscle metabolism during exercise and recovery and may have special applications for studying the generation and transport of acetyl compounds, including acetylcarnitine.