Carbon transfer to milk in Holstein cows in late lactation was measured by introducing changes in the natural stable carbon isotope composition of the feed. Six Holstein cows in mid-lactation were placed on a diet naturally low in 13C (-25.0% vs Pee Dee belemnite [PDB] an international carbon isotope standard), based on alfalfa-barley, and six others were placed on a diet naturally enriched in 13C (-11.5% vs PDB), based on corn. After a 7-wk equilibration period on these diets, three cows were switched from alfalfa-barley to corn, and three were switched from corn to alfalfa-barley. The three other cows in each group served as controls. 13C/12C ratios were measured in daily morning milk samples during the week before and for 6 wk after the changes in diet. After the diets had been switched, milk isotope ratios rapidly approached the isotopic composition of the new diet, indicating rapid transfer of dietary carbon into milk. The data were consistent with a model whereby milk was synthesized from a single precursor pool that responded rapidly to dietary perturbation. The milk precursor pool had a half-life of approximately .9 d and had a mass of approximately 7 kg of carbon, which was renewed daily by the entry of 5 kg of digestible dietary carbon.