To ascertain the activity of the vitamin D biosynthetic pathway, the serum concentration of 1,25-dihydroxyvitamin D (calcitriol) was measured in 16 preterm infants (32 to 37 weeks of gestation) at 1 to 2 and 120 hours of age. Half of the subjects received a daily oral supplement of 2,100 IU of vitamin D3 during the five-day study period. In the first two hours of life, all subjects were hypocalcemic (8.2 +/- 0.2 mg/dl) and 14 subjects had low concentrations of 25-hydroxyvitamin D (calcidiol, 8 +/- 1 ng/ml). The latter finding probably reflects a mild degree of vitamin D deficiency in the mothers of our subjects. Calcitriol concentrations (42 +/- 3 pg/ml) were comparable to those of older children. At 120 hours of age, the control group had no significant change in calcitriol values, whereas the group supplemented with D3 had a more than threefold increase. There was a positive correlation between the circulating concentrations of calcidiol and calcitriol over the period of the study. The data show that, after 32 weeks of gestation, renal 25-hydroxyvitamin D-1 alpha-hydroxylase activity is present, with the rate of calcitriol synthesis being apparently substrate limited. Early neonatal hypocalcemia is therefore unlikely to be caused by an impairment of vitamin D activation.