The primary translation product of rat liver apoE mRNA was isolated from wheat germ cell-free translation systems. Plasma apoE and the primary translation product migrated similarly on SDS-polyacrylamide gels, had similar partial proteolytic peptide maps, and bound to and coeluted from heparin-Sepharose columns. Comparison of the partial amino acid sequence of the primary translation product with the amino-terminal sequence of plasma apoE indicated that rat apoE is initially synthesized with an 18 amino acid amino-terminal extension. This entire segment was removed cotranslationally by canine microsomes possessing signal peptidase activity. The microsome-processed translation product did not contain an endoglycosidase H-sensitive oligosaccharide, suggesting that rat apoE is O-glycosylated.