The fractional rate of breakdown of myofibrillar protein in rat skeletal muscle was measured during subcutaneous cortisone acetate treatment (10 mg/100 g body weight per day). The daily urinary excretion of 3-methylhistidine divided by the 3-methylhistidine pool of the skeletal muscle was used to determine the fractional breakdown rate of myofibrillar protein. The mean fractional breakdown rate remained within the normal range throughout the first 5 days, but decreased significantly from the 16th day of treatment. When the daily 3-methylhistidine excretion was divided by the creatinine excretion, the rate showed the same trend of change. These results strongly suggest that the loss of myofibrillar protein induced by cortisone administration is not caused by increased breakdown but by decreased synthesis.