Familial hyperproinsulinemia is a genetic abnormality characterized by an increased proportion of proinsulin immunoreactivity in the circulation due to mutations affecting the posttranslational processing of proinsulin. In affected Japanese families, this has been associated with noninsulin-dependent diabetes mellitus or impaired glucose tolerance. A three-generation Caucasian family with hyperproinsulinemia was identified through unexplained hyperinsulinemia in a normal volunteer participating in a metabolic study. High pressure liquid chromatography analysis of fasting plasma revealed a major peak eluting close to the position of proinsulin. Direct sequencing of the proinsulin gene exon 3 showed a heterozygous point mutation (CGT-->CAT) resulting in the substitution of Arg-->His in position 65 (corresponding to the AC cleavage site) in the index case, his mother, and his maternal grandmother. Using specific enzyme-linked immunosorbent assay methods to quantify insulin and proinsulin (including its conversion intermediates), the impact of this mutation on B cell secretion and glucose tolerance was studied. All affected subjects had normal oral glucose tolerance. In the basal state and after oral glucose administration, their proinsulin responses were immense, but intact insulin responses were slightly reduced. However, when calculating insulin bioactivity by assuming 9% activity for mutant Arg65-->His proinsulin, responses in affected subjects were comparable to those in normal subjects. In conclusion, our data demonstrate hyperproinsulinemia in a three-generation Caucasian family due to heterozygous mutant Arg65-->His proinsulin. This was not associated with impaired glucose tolerance. These results suggest that this mutation in the heterozygous state per se does not affect glucose tolerance and that the biological activity of mutant proinsulin contributes to glucose homeostasis in this family. The association of the same mutation with impaired glucose tolerance or diabetes in previous studies may be the result of selection bias or associated conditions (e.g. the genetic background of the kindreds examined).