The effect of insulin on fatty acid and glycerol release was examined in fat cells isolated from the parametrial adipose tissue of starved rats. Insulin reduced the basal rate of both fatty acid and glycerol release and blocked the stimulatory effect of growth hormone and dexamethasone on fatty acid and glycerol release by fat cells. The inhibitory effect of insulin on glycerol release was seen whether the cells were incubated in albumin buffer media in the absence or presence of glucose. As little as 0.1 μU/ml of insulin inhibited the acceleration of fatty acid release by growth hormone and dexamethasone, but 10–100 times as much insulin was required to accelerate glucose metabolism. Insulin reduced the stimulation of lipolysis by small amounts of theophylline and ACTH but the antilipolytic effect of insulin was readily overcome by addition of more theophylline or ACTH. Insulin did not reduce the lipolytic action of glucagon. (Endocrinology78: 773, 1966)