The primary structure of apolipoprotein J (apoJ) was deduced by the combined strategies of protein sequencing and cDNA cloning and sequencing. ApoJ, an apolipoprotein associated with discrete subclasses of high-density lipoproteins, is encoded by a single gene in both the human and mouse genomes. ApoJ is synthesized as a 427 amino acid polypeptide that is posttranslationally cleaved at an internal bond between Arg-205 and Ser-206. The subunits of apoJ are designated apoJ alpha, corresponding to residues 1-205, and apoJ beta, corresponding to resides 206-427. The subunits are associated through disulfide bonds. Analysis of the primary structure of apoJ predicts the existence of amphiphilic helices, which may account for the association of apoJ with lipoproteins, and heparin-binding motifs in both subunits. ApoJ appears to be the human analogue of a rat protein present in high concentrations in the testis, sulfated glycoprotein 2. ApoJ mRNA (1.9 kb) is expressed in all but one tissue examined. The mRNA is present in relatively high levels in brain, ovary, testis, and liver, is less abundant in heart, spleen, lung, and breast, and is absent in T-lymphocytes. ApoJ is unique among previously characterized human apolipoproteins in its structure and tissue distribution.