A full length human serum apolipoprotein AI (apo AI) cDNA clone was isolated from a human liver cDNA library. The EcoRI insertion fragment was cloned into expression vectors pDS5 and pDS12 for in vitro transcription and translation. The primary translation product is correctly translocated and the N-terminal signal sequence of the primary translation product of the wild type apo AI cleaved in the presence of dog pancreatic endoplasmic reticulum (ER) membranes releasing proapo AI. Ala-7 at the C-terminus of the signal sequence and Gln-1 of the prosequence were transposed by site-directed mutagenesis thus mutually exchanging the C-termini Gln-8-Ala-7 of the presequence and Gln-2-Gln-1 of the prosequence. The primary translation product of this mutated preproapo AI cDNA is correctly cotranslationally translocated into the lumen of the ER membranes and remains uncleaved by the signal peptidase. Deletion of the hexapeptide prosequence by site-directed mutagenesis in the preproapo AI cDNA led to a primary translation product which is cotranslationally translocated with processing to the mature apo AI polypeptide. We conclude that neither the proteolytic cleavage of the presequence nor the presence of the prosequence are structurally essential for the cotranslational translocation of apo AI. The amino-acid sequence bordering the cleavage site at the C-terminus of the presequence is without influence for the specificity of the signal peptidase.