31P nuclear magnetic resonance (NMR) saturation transfer has been used to measure enzymatic flux through the creatine phosphokinase reaction in the direction of ATP synthesis in the human forearm muscle flexor digitorum superficialis. Modification of the ratio method for measurement of spin-lattice relaxation (R. Freeman, H.D.W. Hill, and R. Kaptein, J. Magn. Reson. 7, 82(1972]was tested and used to appreciably shorten the duration of the measurement. Under conditions of steady state work intracellular pH decreased slightly by 0.06 units and the spin-lattice relaxation time of phosphocreatine in muscle was unchanged, while flux from phosphocreatine to ATP was 64 +/- 10% of the resting value. This is contrary to the increase in flux of 155% predicted from previous saturation transfer studies carried out in vitro on rabbit skeletal muscle creatine phosphokinase using metabolite concentrations to mimic those in vivo (E.A. Shoubridge, J.L. Bland, and G.K. Radda, Biochim. Biophys. Acta 805, 72 (1984]. This discrepancy could be accounted for by an underestimation of the ADP concentrations to which the enzyme is exposed due to inaccurate assumptions about the total metabolite concentrations, or possibly by compartmentation of creatine phosphokinase and its reactants.