Although exogenous 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] administered via intravenous, intramuscular, and oral routes has been tested for efficacy in preventing parturient hypocalcemia in dairy cows, there are few reports concerning intravaginal administration. For this study, 1,25(OH)2D3 was administered via the bovine vaginal lumen, and subsequent changes in blood chemistry, including levels of 1,25(OH)2D3 and several minerals, were measured to confirm vaginal absorption. Each of 5 heifers received a single intravaginal dose of 1 microg of 1,25(OH)2D3/kg body weight; a single control heifer received the ethanol carrier alone. In heifers receiving 1,25(OH)2D3, the plasma 1,25(OH)2D3 levels increased markedly from baseline (88.3 +/- 20.3 pg/mL) within 2 h and reached a peak at 6 h after treatment (1967.4 +/- 1139.6 pg/mL). Plasma Ca levels increased from baseline (10.4 +/- 0.4 mg/dL) to a peak of 11.96 +/- 0.7 mg/dL at 24 h. The levels of inorganic phosphate in plasma increased over time from 7.3 +/- 0.5 to 8.1 +/- 0.8 mg/dL by 6 h and were maintained at a plateau level (9.1 +/- 0.7 to 8.6 +/- 0.6 mg/dL) from 24 to 96 h after treatment. Plasma magnesium decreased from a baseline level of 2.1 +/- 0.1 mg/dL to a plateau level of 1.8 +/- 0.1 mg/dL, which was sustained from 24 to 48 h after treatment. The present study provides evidence of the absorption of exogenous 1,25(OH)2D3 from the bovine vaginal wall, as shown by the marked elevation of plasma 1,25(OH)2D3 levels by 2 h after administration, and indicates the possible utility of intravaginal administration of 1,25(OH)2D3 for prophylaxis of hypocalcemia.