The function of creatine kinase (CK) and its effect on phosphorus metabolites was studied in livers of transgenic mice expressing human ubiquitous mitochondrial CK (CK-Mit) and rat brain CK (CK-B) isoenzymes and their combination. ³¹P NMR spectroscopy and saturation transfer were recorded in livers of anesthetized mice to measure high-energy phosphates and hepatic CK activity. CK reaction velocity was related to total enzyme activity irrespective of the isoenzyme expressed, and it increased with increasing concentrations of creatine (Cr). The fluxes mediated by both isoenzymes in both directions (phosphocreatine or ATP synthesis) were equal. Over a 20-fold increase in CK-Mit activity (28-560 µmol · g wet wt¹ · min¹), the fraction of phosphorylated Cr increased 1.6-fold. Hepatic free ADP concentrations calculated by assuming equilibrium of the CK-catalyzed reaction in vivo decreased from 84 ± 9 to 38 ± 4 nmol/g wet wt. Calculated free ADP levels in mice expressing high levels of CK-B (920-1,635 µmol · g wet wt¹ · min¹) were 52 ± 6 nmol/g wet wt. Mice expressing both isoenzymes had calculated free ADP levels of 36 ± 4 nmol/g wet wt. These findings indicate that CK-Mit catalyzes its reaction equally well in both directions and can lower hepatic apparent free ADP concentrations.