The genes for two of the proteins of the plasma lipid transport system, apolipoprotein AI (apoAI) and CIII (apoCIII) are closely linked in the human genome. An approximately equal to 30-kilobase (kb) DNA segment containing these genes and their flanking sequences has been cloned and extensively characterized. Hybridization studies revealed that a DNA fragment located 12 kb 3' to the apoAI gene contains sequences homologous to a 1.8-kb mRNA transcript in human fetal intestine and adult liver but not in fetal liver, kidney, heart, brain, or muscle. This DNA fragment was used as a probe to isolate a clone from an adult human liver cDNA library. The nucleotide sequence of this clone is 74.8% homologous to the cDNA sequence of rat apolipoprotein AIV (apoAIV), another protein of the lipid transport system, and codes for a protein that is 58.6% identical to rat apoAIV. These results indicate that apoAI, apoCIII, and apoAIV genes are closely linked in the human genome and suggest that all three of them are derived from a common ancestral precursor.