The relationship between the percentage of rod-shaped rat heart myocytes and ATP, creatine phosphate, creatine and inorganic phosphate content was determined. With these values the free energy of ATP hydrolysis was calculated and found to be 59.2 kJ/mol, a much higher value than found for the perfused rat heart. When, during the isolation procedure, creatine was present in the perfusion medium during the low-calcium period, the total creatine content of the myocytes after isolation was comparable to that found in the perfused rat heart. However, when creatine was absent during this low-calcium perfusion period, total creatine content of the myocytes was significantly lower. This difference is caused by leakage of creatine from healthy cells. The free energy of ATP hydrolysis was not affected by the absence of creatine during the low-calcium perfusion period.