Abstract Several semisynthetic analogues of human insulin were prepared by enzyme-assisted coupling of synthetic octapeptides to the C-terminal of porcine desoctapeptide insulin. We report the receptor-binding and biological properties of [LeuB24]- and [LeuB25]-insulins, one of which has the same sequence as a “mutant” insulin recently found in a diabetic patient (Tager, H. et al.(1979) Nature 28 :121–125). [LeuB24]- and [LeuB25]-insulins had, respectively, 8–12% and 0.9–1.1% of the binding affinity of human insulin, and 11% and 2.7% of its potency in stimulating lipogenesis in isolated rat fat cells. Neither one was an antagonist of the biological effects of native insulin. While the ability of [LeuB24]-insulin to induce negative cooperativity was clearly impaired, that of [LeuB25]-insulin was almost abolished. [LeuB25]-insulin was also a potent antagonist of the negative cooperativity induced by native insulin.