T he recently discovered hormone precursors, pork and beef proinsulins, their respective connecting peptides, and beef proinsulin intermediates have been compared to insulin in their ability to stimulate the conversion of glucose-U-¹⁴C to ¹⁴CO₂ and lipids in isolated fat cells. The concentrations of beef and pork proinsulins required to achieve the same biological effect were respectively, 15 and 10 times that of insulin. Beef proinsulin intermediates required only 2.6 times the concentration of insulin for the same effect. Pork and beef connecting peptides in high or low concentrations alone or in combination with proinsulin, insulin, or proinsulin intermediates showed no biological effect on the isolated fat cell system. The insulin-like activity of beef and pork proinsulins on the isolated fat cell system was not abolished with pancreatic trypsin or kallikrein inhibitors. Pork insulin antiserum inhibited the biological activity of pork insulin and proinsulin as well as that of beef insulin or proinsulin. Pork proinsulin antiserum also inhibited the insulin-like activity of both pork insulin and proinsulin. By the radioimmunoassay method, pork insulin antiserum bound only ¼ to [unk] as much proinsulin as insulin. Beef proinsulin intermediates, on the other hand, were found to react with the pork insulin antiserum to an extent nearly equal to that of insulin. These data suggest that (a) proinsulin exhibits its effect on the isolated fat cells independent of its conversion to insulin, (b) connecting peptides have no biological effect under present experimental conditions, and (c) in comparison to insulin, immunological reactivity of proinsulin is greater than its biological activity using our pork insulin antiserum; thus, the comparison of antibody specificity with the fat cell receptor specificity suggests that the biological site of action is different from the immunologic site.