The biosynthesis of the Semliki Forest virus (SFV) structural proteins provides an interesting model system to study the reinitiation of translocation of membrane proteins into the endoplasmic reticulum membrane. The two transmembrane spike proteins, p62 and E1, are derived from a single polypeptide precursor. Once the first protein, p62, has been anchored and its cytoplasmic tail has been synthesized, translocation must be reinitiated to account for the insertion of the E1 protein. We have used deletion mutagenesis of the SFV cDNA to investigate the requirements for this event and map in detail the location of the signal. We have shown by deleting the region encoding the p62 signal and expressing the modified cDNA in COS cells that the p62 protein is not involved in the translocation of the E1 protein. The E1 signal was precisely mapped by progressive truncations of the 6 K peptide (located between p62 and E1 in the SFV polyprotein) and subsequent analysis in cell-free systems. A segment within the last 26 residues of the 6 K peptide was shown to be essential for translocation. This segment was then fused to the N-terminus of the chimpanzee alpha-globin and was shown to be sufficient for translocation. The E1 signal was cleaved efficiently even when attached to the alpha-globin protein. The activity of the signal was found to be SRP dependent in a wheat-germ cell-free system. We conclude that prior attachment of the ribosome to the membrane via the p62 signal peptide is not necessary for E1 translocation and that the reinitiation of translocation is mediated by an independent internal signal likely to be SRP dependent.