The effects of increased ruminal supply of butyrate on milk yield, milk composition, and blood metabolites were studied in four lactating cows in a 4 x 4 Latin square design. The basal diet comprised grass silage, hay, and concentrate (34:22:42, DM basis) and was supplemented with isoenergetic VFA infusions (3.58 Mcal/d). A 3:1 molar mixture of acetate and propionate was replaced gradually with butyrate at the rates of 0, 200, 400, or 600 g/d. When the amount of infused butyrate increased, isobutyrate, butyrate, and isovalerate in plasma and acetoacetate and beta-hydroxybutyrate in whole blood increased linearly, but plasma glucose concentration decreased. The latter was associated with a trend toward higher plasma urea concentration, suggesting that more AA were used for gluconeogenesis as the supply of propionate decreased and that of butyrate increased. Milk yield was not changed. The concentrations of milk fat and protein increased, and that of lactose decreased linearly, with the rate of butyrate infusion. Milk fat yield increased, and lactose yield tended to decrease, with increased butyrate infusion. These results indicate that changes in the supply of butyrate do not affect markedly milk yield in cows yielding less than 20 kg/d but cause marked changes in milk composition. The increase in ruminal butyrate supply increased ketogenesis and decreased gluconeogenesis in the liver of lactating dairy cows.