We evaluated urinary calcium excretion in 21 premature infants fed either a formula containing high concentrations of calcium (Ca) and phosphorus (P) or human milk fortified with a commercially available human milk fortifier. Dual-tracer stable-isotope Ca absorption studies were performed on all infants. Urinary Ca excretion was not significantly related to Ca or P intake or true Ca absorption. The recovery of the orally administered tracer in the urine was used to evaluate the source of calciuria in study subjects. In almost all subjects, tissue-derived (Vbu), rather than diet-derived (Vou), Ca was the principal source of urinary Ca. Hypercalciuric subjects demonstrated greater Vbu and Vou than nonhypercalciuric subjects. Our data demonstrate that moderate hypercalciuria is common in premature infants whose diets are high in mineral content and that hypercalciuria is not related to inadequate mineral intake or Ca absorption but is related, instead, to losses of both tissue and dietary Ca.