The polymorphism of apolipoprotein E (apo E) accounts for a substantial amount of the genetic variance of cholesterol levels in man. The epsilon-2 allele and the epsilon-4 allele raises plasma and low density lipoprotein cholesterol levels as compared to the epsilon-3 allele. Whereas the lower cholesterol levels in carriers of the epsilon-2 allele can, at least in part, be attributed to the grossly deficient binding of apo E-2 to the apo B,E receptor, apo E-3 and E-4 bind to the same degree. We used gel filtration and ultracentrifugation to separate lipoproteins and subsequent immunoblotting analysis to study the apo E isoform distribution. We always found lipoproteins of lower density relatively enriched in apo E-4 and high density lipoproteins relatively depleted of apo E-4 as compared to apo E-3. This was also seen in plasma of heterozygous subjects that simultaneously express two apo E isoforms. Also, the apo E-A-II complex was directly shown by immunoblotting. Furthermore, when purified iodinated apo E was incubated with plasma in vitro, apo E-4 also reassociated more with lipoproteins of lower density than apo E-3. We conclude that apo E-3 and apo E-4 have a different lipoprotein particle distribution in vivo. This differential lipoprotein distribution may account for differences in the metabolism between apo E-3 and E-4.