The effect of bST on the metabolism of lipid in adipose tissue was studied using tissue biopsies from lactating cows treated with bST for 8 d. Cows responded to treatment by increasing daily milk yield by 10.9 kg, although net energy intake was not changed. Thus, net energy balance was changed from highly positive to slightly negative (+7.7 to -1.1 Mcal/d). Consistent with these changes in net energy balance, lipogenesis rates were dramatically reduced (97%) in adipose tissue from bST-treated cows. Activities of acetyl-coenzyme A carboxylase (initial and total) and fatty acid synthase were also dramatically decreased. Therefore, for cows in positive energy balance, reduced lipid synthesis in adipose tissue represents a major mechanism whereby bST alters nutrient partitioning to support greater milk synthesis. Treatment with bST had no effect on beta-adrenergic-stimulated lipolysis in adipose tissue explants incubated in vitro with adenosine deaminase. However, bST treatment reduced the ability of adenosine to inhibit lipolysis in adipose tissue, which involved changes in both sensitivity and responsiveness to adenosine. Therefore, the enhanced lipolytic response to catecholamine in vivo with bST treatment relates to relief in the adenosine inhibitory signaling cascade rather than to a direct effect on the stimulatory signaling pathway.