This study investigated the effects of pinealectomy and exercise training on rat adipose tissue metabolism. Pinealectomized (PINX) and sham-operated (CONTROL) adult male Wistar rats were subdivided into four subgroups, including PINX untrained, PINX trained, CONTROL untrained and CONTROL trained. At the end of the training period (8 wk), the rats were killed and peri-epididymal adipocytes were isolated for in vitro insulin-stimulated glucose uptake, conversion of D-[U-14C]-glucose, l-[U-14C]-lactate, [2-14C]-acetate and [1-14C]-palmitate into 14CO2, and insulin binding. Pinealectomy resulted in a significantly decreased insulin-stimulated glucose uptake in adipocytes without affecting insulin-binding capacity. However, in intact control animals only, training promoted a higher baseline glucose uptake in adipocytes. Training influenced the adipocyte ability to oxidize the different substrates: the rates of glucose and palmitate oxidation increased while the rates of lactate and acetate diminished. Nevertheless, these effects of exercise training were not seen in pinealectomized rats. Additionally, an increase in palmitate oxidation was observed in sedentary pinealectomized animals. In conclusion, these data show that the pineal gland alters the patterns of substrate utilization by the adipocyte, in such a way that its absence disrupts the ability to adapt to the metabolic demands evoked by exercise training in rats.