The effects of regular submaximal exercise on dietary protein requirements, whole body protein turnover, and urinary 3-methylhistidine were determined in six young (26.8 +/- 1.2 yr) and six middle-aged (52.0 +/- 1.9 yr) endurance-trained men. They consumed 0.6, 0.9, or 1.2 g.kg-1.day-1 of high-quality protein over three separate 10-day periods, while maintaining training and constant body weight. Nitrogen measurements in diet, urine, and stool and estimated sweat and miscellaneous nitrogen losses showed that they were all in negative nitrogen balance at a protein intake of 0.6 g.kg-1.day-1. The estimated protein requirement was 0.94 +/- 0.05 g.kg-1.day-1 for the 12 men, with no effect of age. Whole body protein turnover, using [15N]glycine as a tracer, and 3-methylhistidine excretion were not different in the two groups, despite lower physical activity of the middle-aged men. Protein intake affected whole body protein flux and synthesis but not 3-methylhistidine excretion. These data show that habitual endurance exercise was associated with dietary protein needs greater than the current Recommended Dietary Allowance of 0.8 g.kg-1.day-1. However, whole body protein turnover and 3-methylhistidine excretion were not different from values reported for sedentary men.