The effects of nutritional state, insulin, and glucagon on lipid mobilization were determined in rainbow trout, Oncorhynchus mykiss. In nutritional state experiments, fish were either fed continuously (except 24 to 36 hr prior to experimentation) with commercial trout chow or fasted for 4 weeks. Lipase activity in liver tissue isolated from fasted fish and cultured for 5 hr was greater than that in tissue isolated from fed fish and cultured. The presence of glucose (5.55 mM) in the incubation medium accentuates lipolytic activity in both liver and adipose tissue. Hormone response was assessed both in vivo and in vitro. Salmon insulin was injected into anesthetized fish (fed continuously except 24 hr prior to injections) in 10 μl of saline/g body weight; final hormone dose was 100 ng/g body weight. Tissue and plasma were sampled 1 and 3 hr after injection. Insulin resulted in depressed plasma FA concentration and reduced hepatic triacylglycerol lipase activity. In vitro effects of hormones were evaluated by incubating liver and adipose tissue pieces in Hanks-MEM. Glucagon (bovine/porcine) directly stimulated lipid breakdown in both liver and adipose tissue. These actions were manifested by enhanced FA and glycerol released into the culture medium and by elevated triacylglycerol lipase activity. Insulin (bovine) generally appeared antilipolytic as this agent inhibited glucagon-stimulated lipase activity and glucagon-stimulated FA release. Furthermore, insulin (in the presence of glucose) reduced net lipolysis, as indicated by glycerol release, compared to control cultures. These results indicate that nutritional state and glucose are important modulators of lipid mobilization and that glucagon and insulin act directly on lipid storage sites to coordinate lipolysis in rainbow trout.