Vitamin D status increases during healthy mammalian pregnancy, but the molecular determinants remain uncharacterized. The first objective of this study was to determine the effects of pregnancy, and the second objective was to examine the role of chronic hypoxia on vitamin D status and metabolism in an ovine model. We analyzed the plasma levels of cholecalciferol, 25-OH-D, and 1␣,25-(OH)2D in nonpregnant ewes, near-term pregnant ewes, and their fetuses exposed to normoxia (low altitude) or hypoxia (high-altitude) for 100 days. Hypoxic sheep had increased circulating levels of 25-OH-D and 1␣,25-(OH)2D compared with normoxic sheep. Hypoxia increases in 25-OH-D were associated with increased expression of renal 25-hydroxylases CYP2R1 and CYP2J. Pregnancy did not increase further the plasma levels of 25-OH-D, but it significantly increased those of the active metabolite, 1␣,25-(OH)2D, in both normoxic and hypoxic ewes. Increased bioactivation of vitamin D correlated with increased expression of the vitamin D-activating enzyme CYP27b1 and decreased expression of the inactivating enzyme CYP24a1 in maternal kidneys and placentas. Hypoxia increased parathyroid hormone levels and further increased renal CYP27b1. Pregnancy and hypoxia decreased the expression of vitamin D receptor (VDR) in maternal kidney and lung, with opposite effects on placental VDR. We conclude that ovine pregnancy is a model of increased vitamin D status, and long-term hypoxia further improves vitamin D status due to pregnancy-and hypoxia-specific regulation of VDR and metabolic enzymes.pregnancy; hypoxia; vitamin D; metabolism; kidney; placenta VITAMIN D HAS WELL-ESTABLISHED CLASSIC EFFECTS on bone metabolism and mineral homeostasis (8,33). In addition, vitamin D has important noncalcemic roles in the reproductive, cardiovascular, renal, lung, immune, neuronal, and pancreatic systems (8, 33). In reproduction, vitamin D deficiency has been associated with sterility, placental insufficiency, intrauterine growth restriction, recurrent miscarriages, gestational diabetes, and preeclampsia (maternal high blood pressure and proteinuria) (6, 51-53). Furthermore, maternal vitamin D deficiency has been also linked to postnatal infectious diseases, autoimmune diseases (diabetes type I), asthma, and obesity (26,28,54).Vitamin D 3 (cholecalciferol) can be produced endogenously from 7-dehydrocholesterol by sun exposure in the skin epidermis or obtained from dietary sources (8,33 (14). A classic gene regulated in this manner is the vitamin D-inactivating enzyme CYP24A1 (24-hydroxylase). Most cells that express VDR also express CYP24A1, providing a unique negative feedback control mechanism to regulate the vitamin D effects (8,14). In healthy nonpregnant mammals, the circulating levels of 1␣,25-(OH) 2 D are tightly regulated; low levels of 1␣,25-(OH) 2 D stimulate the release of parathyroid hormone (PTH) that upregulates renal CYP27b1 expression, and high levels of 1␣,25-(OH) 2 D stimulates VDR that upregulates CYP24a1 expression (8, 33).The metabolism of vitamin ...