Upon isoelectric focusing, the apolipoprotein E (apoE) system in man demonstrates at least five subspecies. The system constitutes a complex polymorphism that gives rise to five genetically determined phenotypes. Patients with type III hyperlipoproteinemia are markedly deficient in the apoE-3 subspecies. In order to determine whether the various phenotypes differ with respect to the total amount of protein present, we measured the apoE levels in plasma by RIA in six families ascertained through a well documented type III proband. ApoE-3-deficient homozygotes were found to have significantly more protein than heterozygotes who, in turn, had significantly more protein than normal homozygotes. These differences remained significant after allowance was made for the correlated effects of very low density lipoproteins cholesterol and very low density lipoproteins triglycerides. Among heterozygotes, persons with the fifth isoelectric focusing band (apoE-4) were found to have significantly less apoE protein than heterozygotes without this band. A similar but nonsignificant trend was observed in normal homozygotes. These observations are consistent with the hypothesis that the primary defect in type III hyperlipoproteinemia subspecies to another.