To assess the influence of protein intake on renal excretion of calcium and amino acids and on bone mineralization in preterm infants, we randomly selected within weight group strata 27 infants who weighed < 1500 gm at birth (nine per group) to be fed a high-mineral (calcium, 940 mg/L; phosphorus, 470 mg/L) premature formula with one of the following protein contents: formula A, 3.0 gm/100 kcal; formula B, 2.7 gm/100 kcal; and formula C, 2.2 gm/100 kcal. Mean (+/- SD) daily weight gain was greater in infants receiving the higher protein intakes for the first 30 days (formula A, 24.8 +/- 5.1 gm; formula B, 20.5 +/- 3.8 gm; formula C, 16.2 +/- 5.9 gm (analysis of variance: p < 0.01; C < A, p < 0.05)). Bone mineral content did not differ at any time point, and all groups had a high prevalence of generalized aminoaciduria (4 weeks: formula A, 56%; formula B, 71%; formula C, 75%). Urinary calcium corrected for creatinine (in milligrams per milligram) increased as protein content decreased (2 weeks: formula A, 0.16 +/- 0.10; formula B, 0.20 +/- 013; formula C, 0.44 +/- 0.33 (C > A, C > B, p < 0.05); 4 weeks: formula A, 0.23 +/- 0.15; formula B,0.34 +/- 0.47; formula C, 0.49 +/- 0.22 (C > A, p < 0.01). We conclude that the high mineral content and other components of premature formulas result in a higher growth rate and may increase protein requirements. Failure to meet protein requirements may result in underutilization of absorbed calcium and increased renal excretion of calcium. In preterm infants, higher protein intake probably supports rather than jeopardizes bone mineral accretion, and reduces rather then increases calciuria.