We have characterized the NH2-terminal sequence of the primary translation product of intestinal apolipoprotein A-I mRNA. Co-translational cleavage of this in vitro product and NH2-terminal sequence analysis of plasma high density lipoprotein-associated apolipoprotein A-I showed that it is initially synthesized as a preproprotein. The 24-amino-acid NH2-terminal extension consists of an 18-amino-acid presegment; Met-Lys-Ala-Ala-Val-Leu-Ala-Val-Ala-Leu-Val-Phe-Leu-Thr-Gly-Cys-Gln-Ala, and a hexapeptide prosegment; -X-Glu-Phe-X-Gln-Gln, followed by the NH2 terminus of the "mature" plasma protein: Asp-Glu-Pro-Gln-X-Gln-Trp-Asp-Arg-Val-Lys-Asp-Phe-Ala-X-Val-Tyr-X-Asp-Ala-Val. Although the prepeptide resembles other signal peptides, the prosegment differs from other propeptides in that it does not terminate with paired basic amino acid residues. These results suggest that translocation of nascent preproapolipoprotein A-I is similar to that of other secretory proteins. However, post-translational proteolytic processing of proapolipoprotein A-I must be unique, and could play a role in the formation of nascent high density lipoprotein particles.