Adipose triglyceride lipase (ATGL) catalyzes the rate-limiting removal of the first fatty acid from a triglyceride. ATGL is activated by comparative gene identification-58 (CGI-58) and inhibited by G(0)/G(1) switch gene-2 protein (G0S2). Research in other tissues and cell culture indicates that inhibition is dependent on relative G0S2-to-ATGL protein content. G0S2 may also have several roles within mitochondria, however this has yet to be observed in skeletal muscle. The purpose of this study was to determine if muscle G0S2 relative to ATGL content would decrease to facilitate intramuscular lipolysis following endurance training. Male Sprague-Dawley rats (n = 10, starting age 51-53 days) were progressively treadmill trained at a 10% incline for 8-weeks ending with 25m/min for 1 hour, compared to control. Sciatic nerve stimulation for hind limb muscle contraction (and lipolysis) was administered for 30 minutes to one leg, leaving the opposing leg as a resting control. Soleus (SOL), red (RG) and white gastrocnemius (WG) were excised from both legs following stimulation or control. ATGL protein increased in all trained muscles. Unexpectedly, G0S2 protein was greater in the trained SOL and RG. In RG isolated mitochondria, G0S2 also increased with training, yet mitochondrial G0S2 content was unaltered with acute contraction, therefore any role of G0S2 in the mitochondria does not appear to be acutely mediated by content alone. In summary, G0S2 increased with training in oxidative muscles and mitochondria but not following acute contraction, suggesting that inhibition is not through relative G0S2-to-ATGL content, but through more complicated intracellular mechanisms.