A dual-tracer, stable-isotope method was used to measure the percentage of true calcium absorption (alpha), true calcium absorption rate (Va), endogenous fecal calcium excretion rate (Vf), urinary calcium excretion rate (Vu), and calcium retention rate (Vr). Twenty-seven infants with a mean gestation of 30.6 +/- 1.7 weeks and a mean birth weight of 1.4 +/- 0.21 kg were studied at 2 or 3 weeks of age, or both, during feedings of human milk (HM), fortified human milk (HMF), and commercially available formula (20 or 24 calories per ounce) for premature infants (EPF-20/780 and EPF-24/940) (part 1 of our study). Of 13 additional infants with a mean (+/- SD) birth weight of 1.26 +/- 0.25 kg and gestation of 29.6 +/- 2.5 weeks, 11 completed a crossover-design study at 2 and 3 weeks of age, receiving two identical formulas containing calcium, 940 mg/L, and phosphorus, 470 mg/L (EPF-24/940 formula) or calcium 1340 mg/L, and phosphorus, 680 mg/L (EPF-24/1340 formula) (part 2 of our study). The alpha value was higher in infants receiving HM (76.4 +/- 15.1%) or HMF (68.0 +/- 7.8%) than in those receiving EPF-20/750 formula (54.1% +/- 5.6%) or in previously reported infants fed standard formula (47.1% +/- 11.5%); those given EPF-24/940 formula had intermediate values (63.9% +/- 13.9%, part 1; 56.1% +/- 16.5%, part 2). No significant differences existed among groups for either Vu or Vf per kilogram. In the crossover study (part 2), no significant differences were seen between formulas for alpha and for Va, Vf, or Vr per kilogram. However, Vu per kilogram was significantly decreased in infants receiving the higher mineral formula (EPF-24/940: 3.6 +/- 2.3; EPF-24/1340: 2.9 +/- 2.3 mg/kg per day; p = < 0.005). With all feedings, alpha, Vu per kilogram, and Vf per kilogram were not related to gestational age, age at study, calcium intake, or each other. However, Vf per kilogram was inversely related to birth weight. Thus, alpha, Vu, and Vf appear to be independent and may be differentially affected by factors altering calcium dynamics. We conclude that increasing formula mineral content does not ensure increased retention; careful monitoring of individual infants remains indicated.