Leptin secretion by adipose tissue is involved in many physiological control systems, including those that determine growth, development, body composition, milk production, and reproductive function. In the adipocyte of monogastric animals, malonyl CoA (coenzyme A) seems to link the flux of energy substrates to the control of leptin production. In this study, we tested this for ruminants by examining the effect of cerulenin, an inhibitor of de novo fatty acid synthesis at the step from malonyl CoA to palmitate, on leptin production by cultured bovine adipocytes derived from intermuscular fat. Purified preadipocytes were obtained by the ceiling culture method, and adipogenic media were used to induce their differentiation into adipocytes. We found that leptin concentrations increased significantly with time in culture, and with increases in glucose concentration. Addition of 2-deoxy-D-glucose to the medium, a competitive inhibitor of glucose transport and metabolism, suppressed leptin secretion. In media with high glucose concentrations, cerulenin enhanced leptin secretion. We conclude that, as in monogastrics, malonyl CoA may play a key role in the control of leptin secretion in ruminants.