The current study investigated the effects of chronic training and pinealectomy on the lipogenic and lipolytic activity of adipose tissue. Pinealectomized and sham-operated adult male Wistar rats were distributed in to four subgroups: pinealectomized untrained, pinealectomized trained, control untrained and control trained. At the end of the training period (8 wk) the rats were killed. Blood samples were collected for glucose, insulin and leptin determinations. Peri-epididymal adipocytes were isolated for measurement of in vitro rates of lipolysis and incorporation of substrates (D-[U-14C]-glucose, L-[U-14C]-lactate, [2-14C]-acetate and [1-14C]-palmitate) into lipids, and samples of epididymal adipose tissue were homogenized for evaluation of glucose-6-phosphate dehydrogenase maximal activity. Pinealectomy resulted in a significantly increased lipolytic capacity in response to isoproterenol and a decrease in circulating leptin levels without affecting the rates of incorporation of different substrates into lipids. However, only in the intact control group did training promote a higher basal and isoproterenol-stimulated lipolysis, increase the incorporation of palmitate (esterification), decrease the incorporation of acetate (lipogenesis) into lipids and diminish circulating leptin levels. These effects of exercise training were not seen in pinealectomized rats. However, pinealectomized trained animals showed a marked reduction in lipolysis and an increased rate of acetate incorporation. In conclusion, we demonstrated for the first time that the pineal gland plays an important role in the regulation of lipid metabolism in such a way that its absence caused a severe alteration in the balance between lipogenesis and lipolysis, which becomes evident with the adaptation to exercise training.