We have identified a 65-year-old non-obese Japanese man with diabetes mellitus, fasting hyperinsulinemia (25-50 μU /ml), and a reduced fasting C-peptide/insulin molar ratio of 2.5-3.0.Fastinghyperinsulinemia was also found in his son and daughter. The patient responded normally to exogenous insulin. To define the basis of this disorder, the insulin gene of the patient was amplified by the polymerase chain reaction and the products were sequenced. A novel point mutation was identified in which guanine was replaced by thymine in the insulin gene. The resulting substitution of leucine for arginine at amino-acid position 65 gave rise to a new Hind-III recognition site. The replacement of Arg-65 by Leu also prevents recognition of the C-peptide-A-chain dibasic protease and results in elevation of a proinsulin like-material in the circulation. Furthermore, in this family the proinsulin like-material is the result of a biosynthetic defect inherited as an autosomal dominant trait.