Metabolic problems related to negative energy balance suggest a role for the balance in supply of lipogenic and glucogenic nutrients. To test the effect of lipogenic and glucogenic nutrients on energy partitioning, energy balance and nitrogen balance of 16 lactating dairy cows were determined by indirect calorimetry in climate respiration chambers from wk 2 to 9 postpartum. Cows were fed a diet high in lipogenic nutrients or a diet high in glucogenic nutrients from wk 3 prepartum until wk 9 postpartum. Diets were isocaloric (net energy basis) and equal in intestinal digestible protein. There was no effect of diet on metabolizable energy intake and heat production. Cows fed the lipogenic diet partitioned more energy to milk than cows fed the glucogenic diet [1,175 +/- 18 vs. 1,073 +/- 12 kJ/(kg(0.75) x d)] and had a higher milk fat yield (1.89 +/- 0.02 vs. 1.67 +/- 0.03 kg/d). The increase in milk fat production was caused by an increase in C16:0, C18:0, and C18:1 in milk fat. No difference was found in energy retained as body protein, but energy mobilized from body fat tended to be higher in cows fed the lipogenic diet than in cows fed the glucogenic diet [190 +/- 23 vs. 113 +/- 26 kJ/(kg(0.75) x d)]. Overall, results demonstrate that energy partitioning between milk and body tissue can be altered by feeding isocaloric diets differing in lipogenic and glucogenic nutrient content.