To study the dependence of the forward flux of creatine kinase (CK) on its substrates and products we designed an acute normoxic model of steady-state depletion of phosphocreatine (PCr) and adenylate in the isovolumic acetate-perfused rat heart. Various concentrations of PCr and ATP were induced by prior perfusion with 2 deoxy-D-glucose in the presence of insulin. The apparent rate constant (k(f)) and the forward CK flux were measured under metabolic and contractile steady state by progressive saturation-transfer 31P nuclear magnetic resonance (NMR). At high adenylate content CK flux was constant for a twofold reduction in PCr concentration ([PCr]); CK flux was 6.3 +/- 0.6 mM/s (vs. 6.5 +/- 0.2 mM/s in control) because of a doubling of k(f). Although, at the lowest ATP concentration and [PCr], CK flux was reduced by 50%, it nevertheless always remained higher than ATP synthesis estimated by parallel oxygen consumption measurement. NMR-measured flux was compared with the flux computed under the hypothesis of CK equilibrium. CK flux could not be fully predicted by the concentrations of CK metabolites. This is discussed in terms of metabolite and CK isozyme compartmentation.