The purpose of the present study was to investigate the effect of acute exercise on lipolysis via coordination of hormone-sensitive lipase (HSL) and scaffold proteins, i.e., perilipin A and comparative gene identification-58 (CGI-58), in rat primary adipocytes. Glycerol release was significantly elevated immediately (0 h) and three hours (3 h) after exercise. Both activity and localization to the pellet of HSL were significantly greater in the pellet fraction, which is included in lipid droplet associated-proteins, than in the supernatant fraction. In the pellet fraction, although neither perilipin A nor CGI-58 protein level changed, level of perilipin A/CGI-58 complex was significantly reduced, accompanied by up-regulated association of perilipin A/HSL at 0 h and 3 h after exercise. On the other hand, there were no changes in these molecules at 24 h after exercise, despite a significant decrease in lipolysis that was observed in response to isoproterenol. These findings suggest that acute exercise enhances lipolysis up to at least 3 h after exercise in a manner dependent on modification of HSL and its association with and alteration in scaffold protein. (C) 2010 Elsevier Inc. All rights reserved.