We investigated the mechanism and characteristics of 25-hydroxyvitamin D3 (25-OH-D3) absorption in the unanesthetized rat by using a single-pass intestinal perfusion technique. The rate of 25-OH-D3 absorption remained linear for a wide range of concentrations (2-900 nM). Absorption rate of 25-OH-D3 increased as the pH, the bile acid concentration, and thickness of the unstirred water layer were decreased. Absorption did not change after the additions of fatty acids of varied chain lengths and degrees of saturation. In rats with lymph and bile fistulas, 18.5% and 16.3% of the infused radio-activity appeared in the lymph and bile drainage, respectively. These experiments indicate that 25-OH-D3 is absorbed by a passive diffusion mechanism that is influenced by the intestinal pH, bile acid concentration, and thickness of the unstirred water layer, but not by the presence of fatty acids. Approximately equal fractions of the infused hydroxylated vitamin are recovered from the lymphatic and biliary fluids.