Vitamin D is critical to calcium and phosphorus homeostasis and skeletal health through the life span and may also influence extraskeletal health. Low maternal serum 25-hydroxyvitamin D (25(OH)D) concentration during pregnancy has been associated with many adverse outcomes including preeclampsia and poor neonatal bone health. However, questions remain about the functions of vitamin D during human pregnancy and gestation. Pregnancy is associated with adaptations in maternal vitamin D and calcium metabolism, and these adaptations alter indicators of vitamin D function. As a result, evaluation of serum 25(OH)D concentration and other vitamin D biomarkers is complex and may differ during pregnancy. The goal of this dissertation was to improve evaluation of vitamin D status and understanding of vitamin D metabolism during pregnancy. Two approaches were used: 1) a study of candidate biomarkers in pregnant adolescents and 2) a study of vitamin D3 kinetics in nonpregnant and pregnant women. Promising candidate biomarkers of vitamin D status include the serum 24,25(OH)2D concentration, which is an indicator of vitamin D catabolism, and the measured free 25(OH)D concentration. In study 1, we longitudinally assessed a comprehensive suite of vitamin D biomarkers including serum 24,25(OH)2D, free 25(OH)D, and vitamin D binding protein (DBP) in pregnant adolescents. We found that gestational age and maternal serum 25(OH)D concentration interacted to affect the 24,25(OH)2D concentration. Our results suggest that assessment of 24,25(OH)2D may provide information about the adequacy of the 25(OH)D supply during pregnancy. Furthermore, longitudinal increase in DBP was associated with modest decrease in measured free 25(OH)D. However, free 25(OH)D provided no advantage over total 25(OH)D as a predictor of circulating markers of vitamin D and calcium metabolism. In study 2, we developed a protocol and analytical methods to assess the serum appearance and disappearance of stable isotope-labeled vitamin D3 and labeled 25(OH)D3 after a single oral dose of trideuterated vitamin D3. Overall, the serum tracer concentration-time curves suggest our technique is a valid new tool for investigating vitamin D metabolism across physiologic conditions. Serum DBP concentration was higher in pregnant compared with nonpregnant women and was the main predictor of the area under the curve of trideuterated 25(OH)D3.