We investigated the relationship between serum total and free 1,25-dihydroxyvitamin D (1,25-OH2D) and the biochemical regulation of 1,25-OH2D production in premature infants. We measured 1,25-OH2D, vitamin D binding protein and related biochemical parameters and calculated the free 1,25-OH2D index in serum of 17 premature infants (birthweight 810-1700 g; gestational age 31-36 weeks) on two different occasions defined by body weight (Study A: 1,750-1,850 g, Study B: 2,100-2,200 g). Dietary calcium (Ca) intake was 1,5 or 2,6 mmol/kg/d, phosphorus (P) intake 1,7 mmol/kg/d and vitamin D intake 1,000 IU/d. Biochemical results were similar in infants with different Ca intakes and all were within reference ranges. Concentrations of vitamin D binding protein (Study A 0.15 +/- 0.03 g/l, Study B 0.14 +/- 0.03 g/l; means +/- SD) were lower, concentrations of 1,25 (OH)2D (Study A 180 +/- 67 pmol/l, Study B 216 +/- 53 pmol/l) were higher, and consequently the free 1,25-OH2D index (Study A 6.6 +/- 2.7, Study B 8.8 +/- 2.6) was 4 to 6 times higher than in previously studied term infants. 1,25-OH2D and the free 1,25-OH2D index increased significantly with age and were not correlated with serum P or parathyroid hormone. The data indicate that in premature infants with normal biochemical parameters of Ca and P metabolism elevated concentrations of 1,25-OH2D signify an increased fraction of free 1,25-OH2D and that increased production of 1,25-OH2D is not due to hypophosphatemia or hyperparathyroidism.