Short-term creatine supplementation is reported to result in a decreased ratio of phosphocreatine (PCr) to total creatine (TCr) in human skeletal muscle at rest. Assuming equilibrium of the creatine kinase reaction, this decrease in PCr:TCr implies increased cytoplasmic ADP and decreased Gibbs free energy of ATP hydrolysis in muscle, which seems contrary to the reported ergogenic benefits of creatine supplementation. This study measured changes in PCr and TCr in vastus lateralis muscle of adult men (N = 6, 21-35 y old) during and 1 day after 5 d of creatine monohydrate supplementation (0.43 g.kg body weight(-1).d(-1)) using noninvasive 31P and 1H magnetic-resonance spectroscopy (MRS). Plasma and red-blood-cell creatine increased by 10-fold and 2-fold, respectively, by the third day of supplementation. MRS-measured skeletal muscle PCr and TCr increased linearly and in parallel throughout the 5 d, and there was no significant difference in the percentage increase in muscle PCr (11.7% +/- 2.3% after 5 d) vs. TCr (14.9% +/- 4.1%) at any time point. The results indicate that creatine supplementation does not alter the PCr:TCr ratio, and hence the cytoplasmic Gibbs free energy of ATP hydrolysis, in human skeletal muscle at rest.