Shaw and Chance have recently demonstrated that Kunitz pancreatic trypsin inhibitor (KPTI) blocks the increased conversion of labeled glucose to carbon dioxide and fatty acids in normal adipose tissue of rats in the presence of porcine proinsulin. The experiments reported here were designed to observe the antilipolytic effect of insulin and porcine proinsulin on lipolysis induced by epinephrine and glucagon in isolated fat cells and intact fat pads from rats, both in the presence and absence of KPTI and soybean trypsin inhibitor. It was found that both insulin and proinsulin are potent inhibitors of lipolysis induced by DL-arterenol and by glucagon. Neither KPTI nor soybean trypsin inhibitor blocked the antilipolytic effect of insulin or proinsulin when lipolysis was stimulated by DL-arterenol or by glucagon. The failure of KPTI to inhibit the antilipolytic effect of proinsulin on isolated fat cells may have been caused by inactivation of KPTI by collagenase used in preparation of the isolated fat cells. To test this hypothesis experiments were performed using intact fat pads. As with the isolated fat cells, KPTI did not inhibit the antilipolytic effect of insulin or proinsulin. These observations indicate that, in isolated fat cells and intact fat pads of the rat, proinsulin itself is an effective antilipolytic agent.