Mammary transfer of label from intraperitoneally injected 50 microCi [1 alpha, 2 alpha(n)-hydrogen-3] cholecalciferol, and 50 microCi (26,27-methyl-hydrogen-3)cholecalciferol was studied in nursing rabbits. Does were injected at 3 days postpartum with one of the two labeled compounds. Pups were killed at either 1, 2, 3, 4, or 5 days after dosing of the does, and does were killed after 5 days. Concentrations of radioactivity were greater in tissues of does dosed with tritiated vitamin D3 than in tissues of those dosed with tritiated 25-hydroxyvitamin D3. Concentrations of radioactivity were greater in maternal tissues than in tissues of pups. On the 5th day following administration of tritiated vitamin D3 or 25-hydroxyvitamin D3, the major portion of the radioactivity in does plasma and liver was associated with tritiated 25-hydroxyvitamin D3. In pups from the tritiated vitamin D3 group, the concentration of plasma radioactivity associated with 25-hydroxyvitamin D3 (isolated by high pressure liquid chromatography) increased significantly with time, reaching 85% of the total vitamin D and metabolite radioactivity in the pups at the 5th day. Over 90% of the total recovered plasma radioactivity of pups of the tritiated 25-hydroxyvitamin D3 group was associated with the 25-hydroxyvitamin D3. Much more radioactivity was secreted in the milk of tritiated 25-hydroxyvitamin D3 dosed does than in milk of does dosed with tritiated vitamin D3.