In view of recent reports that high protein diets cause a negative calcium balance and may be a factor in osteoporosis in human adults, a study was conducted to determine the influence of dietary protein intake on calcium metabolism in adult rats. Rats were “deep labeled” with 45Ca by intraperitoneal administration of repeated doses of isotope 1 month before the beginning of the experiments. They were then fed diets containing uniform concentrations of calcium and phosphorus (0.6 and 0.3%, respectively) and low, moderate, or high concentrations of protein (10, 20, or 40%). Unlabeled calcium and 45Ca excretions in the urine were markedly increased (P < 0.01) in rats fed 40% protein as opposed to 10 or 20% protein. However, fecal 45Ca excretion decreased with increments in dietary protein, and consequently no increase in total 45Ca excretion (an indicator of bone resorption) was seen in the high protein group. A slightly greater total loss of 45Ca occurred in the 10% protein group than in the other groups, indicating that their diet was mildly suboptimal in protein. The specific activity of urinary calcium decreased as protein intake was increased, reflecting an enhanced absorption of dietary calcium. Increasing dietary phosphorus to 1.2% led to an increase in 45Ca loss of about 50% at all levels of protein; hence, the increase in calcium absorption induced by dietary protein did not counteract the stimulus to bone resorption caused by a high intake of phosphorus. This study indicates that in the rat, the increased excretion of calcium in the urine seen under conditions of high protein intake is due to a shift in the route of excretion of endogenous calcium from the feces to the urine and to an increase in absorption of dietary calcium. A high protein diet appears to have no effect on bone resorption when calcium and phosphorus intakes are adequate.