We investigated the effect of a single bout of exercise on leptin mRNA levels in rat white adipose tissue. Male Sprague-Dawley rats were randomly assigned to an exercise or control group. Acute exercise was performed on a rodent treadmill and was carried out to exhaustion, lasting an average of 85.5 ± 1.5 min. At the end of exercise, soleus muscle and liver glycogen were reduced by 88% (P < 0.001). Acutely exercised animals had lower (P < 0.05) leptin mRNA levels in retroperitoneal but not epididymal fat, and this was independent of fat pad weight. To test the hypothesis that ₃-adrenergic-receptor stimulation was involved in the downregulation of leptin mRNA in retroperitoneal fat, a second experiment was performed in which rats were randomized into one of four groups: control, control + ₃-antagonist, exercise, and exercise + ₃-antagonist. A highly selective ₃-antagonist (SR-59230A) or vehicle was given by gavage 30 min before exercise or control experiment. Exercise consisted of 55 min of treadmill running, sufficient to reduce liver and muscle glycogen by 70 and 80%, respectively (both P < 0.0001). Again, acute exercise reduced leptin mRNA in retroperitoneal fat (exercise vs. control; P < 0.05), but ₃-antagonism blocked this effect (exercise + ₃-antagonist vs. control + ₃-antagonist; P = 0.42). Unexpectedly, exercise increased serum leptin. This would be consistent with the idea that there are releasable, preformed pools of leptin within adipocytes. We conclude that ₃-receptor stimulation is a mechanism by which acute exercise downregulates retroperitoneal adipose tissue leptin mRNA in vivo.