Nine multiparous Holstein cows (average 5.8 yr, 63 to 132 days postpartum) were in a 28-day single reversal experiment in the Beltsville open circuit respiration chambers with two 14-day injection periods (placebo versus bovine growth hormone, 51.5 IU/day). With growth hormone treatment, milk fat percentage increased 25 to 29%, milk fat yield increased 41%, and cows averaged -13.7 Mcal/day energy balance. There were small increases of triglycerides and 1,2-diglycerides, core lipids, and small decreases of cholesterol and phospholipids, the membrane lipids. Fat from growth hormone treatment had 6% less short (6:0, 8:0, 10:0) and medium (12:0, 14:0, 14:1, 15:0, 16:0) chain fatty acids and 6% more long chain 18:1 fatty acids. Plasma of cows treated with growth hormone had an increase of concentrations of free fatty acids, but no shifts were apparent among other lipid classes. Analysis of total plasma fatty acids did not show any net changes, but within individual plasma lipid classes, 18:1 increased and 18:2 decreased. Overall changes of blood and milk lipids are consistent with the concept that adipose tissue reserves were mobilized in response to hormone treatment and that these mobilized lipids were the major carbon source for the 41% increase of milk fat secretion. Increases of de novo synthesis of short and medium chain fatty acids also occurred but much less. Cows treated with growth hormone were in negative energy balance, and the mechanism may differ when cows are in positive energy balance.