Plasma levels of vitamin D3 or 25-hydroxyvitamin D3 in ewes after administration of a single massive intravenous dose of vitamin D3 (2 X 10(6) IU) or 25-hydroxy vitamin D3 (5 mg) were determined at zero, one, two, three, five, ten and 20 days postinjection. In six ewes injected with vitamin D3 conversion of vitamin D3 to 25-hydroxy vitamin D3 resulted in a six-fold increase in the plasma 25-hydroxy vitamin D3 level within one day. Elevated levels were maintained until day 10 but by day 20 a substantial decline in the plasma 25-hydroxy vitamin D3 level had occurred. Peak levels of vitamin D3 were reached one day after injection and then continuously declined until day 20. Administration of 25-hydroxy vitamin D3 increased plasma concentrations of 25-hydroxy vitamin D3 to fivefold higher levels than those observed when vitamin D3 was injected, with approximately threefold higher levels of 25-hydroxy vitamin D3 maintained for five days. On day 10 and day 20 ewes which were injected with 25-hydroxy vitamin D3 still maintained plasma levels of 25-hydroxy vitamin D3 which were twice as high as those of ewes injected with vitamin D3. In six ewes injected with vitamin D3, a sharp increase in vitamin D3 level in milk occurred within one day and more than a tenfold elevation of milk vitamin D3 concentrations were maintained for ten days. By 20 days the milk vitamin D3 level had returned to preinjection levels. These observations suggest that indirect supplementation of the suckling ruminant with vitamin D3 may be achieved through maternal injection and subsequent mammary transfer.