Serum concentrations of 25-hydroxyvitamin D (25OHD) and 24,25-dihydroxyvitamin D (24,25(OH)2D) were measured in a cross-sectional study of 94 normal pregnant women at various stages of gestation in order to assess the hormonal regulation of calcium homeostasis during gestation. The 40 week gestational period was divided into four 10 weeks quarters. 25OHD concentration were significantly below control levels (32.2 +/- 3.1 (S.E.) ng/ml) by the second quarter of pregnancy and were even lower at term. Serum levels of 24,25(OH)2D did not decrease until the fourth quarter, when the mean concentration (0.8 +/- 0.1 ng/ml) was approximately one half the control values (1.5 +/- 0.3, p less than 0.025). These data suggest that the metabolic pathways of vitamin D are altered during gestation, perhaps in response to increasing mother-to-fetus transport of calcium. There is decreased 24-hydroxylation and, in view of the lowered 25OHD levels, possibly increased production of 1,25-hydroxyvitamin D (1,25(OH)2D).