Protein turnover was studied in eight premature infants of conceptual age 26-37 weeks. A stochastic model based upon [15N]urea or [15N]ammonia excretion following a single injection of [15N]glycine was used to estimate rates of whole body protein synthesis and catabolism. The urinary 3-methylhistidine/creatinine ratio was determined to differentiate skeletal muscle protein breakdown from total protein catabolism. The rates of whole body protein synthesis ranged from 5.2 to 13.2 g X kg-1 X day-1 and protein catabolism ranged from 4.1 to 12.4 g X kg-1 X day-1. Linear regression analyses of conceptual age versus (a) whole body nitrogen flux, (b) protein synthesis, and (c) protein catabolism showed significant inverse relationships. A similar relationship obtained between conceptual age and the urine 3-methylhistidine/creatinine ratio. Muscle protein breakdown did not vary with conceptual age, but the fraction of whole body protein breakdown derived from muscle protein breakdown increased significantly with advancing maturation. The ratio net tissue protein gain/total body protein synthesis increased significantly with increasing body weight. Net tissue protein gain appeared to be directly related to protein and caloric intake. The ratio of the rate of whole body protein synthesis and protein intake was greatest in the youngest infants and declined with maturation. A similar relationship was not found between the ratio of protein synthesis and caloric intake and the degree of maturity. More than 90% of nitrogen entering the metabolic pool was used for protein synthesis and more than 50% of calories administered were similarly utilized. We conclude that: (a) protein turnover in premature infants is far more rapid than in term infants, children, or adults and is inversely related to conceptual age; (b) muscle protein turnover constitutes a greater fraction of overall turnover with advancing maturity; (c) energy and protein intake affect net tissue protein gain significantly in rapidly growing infants; and (d) the efficiency of protein synthesis as a function of protein intake is higher in the most immature infants.