The hypoglycemic actions of human insulin (1 IU/kg b.w.) and biosynthetic human proinsulin in about equimolar amounts were studied after intravenous and subcutaneous injection in rabbits. Blood samples were taken up to four hours after injection for the determination of blood glucose and immunoreactive levels of both insulin and human C-peptide. For the determination of human C-peptide, serum taken after proinsulin injection was divided into two fractions. One was examined directly by the human C-peptide radioimmunoassay and the other after incubation with a protein-A-sepharose coupled insulin antibody to find "free human C-peptide". Proinsulin in amounts equimolar to 1 IU insulin/kg b.w., exerted a 34% stronger hypoglycemic action after subcutaneous injection than after intravenous administration (area under curve estimation). Proinsulin-induced hypoglycemia did not last longer after intravenous administration than that induced by intravenous insulin. Although subcutaneous proinsulin did not show the same maximum decrease of blood glucose compared to subcutaneous insulin, its action was significantly prolonged (up to 180 min). Specific measurement of free human C-peptide showed no evidence of conversion of proinsulin to insulin and C-peptide.