Sindbis virus glycoproteins PE2 (precursor of E2) and E1 are coded in this order by the same monocistronic mRNA, cotranslationally inserted in the endoplasmic reticulum membrane and then transported to the cell surface where the progeny virus is released by budding. In the virion, three E1 plus three E2 molecules form hexameric spike complexes. Previous work (S. Bonatti, G. Migliaccio, G. Blobel, and P. Walter (1984), Eur. J. Biochem. 140, 499-502) revealed a single signal sequence for cotranslational translocation located at the aminoterminus of PE2. We have generated progressive deletions of the coding region upstream of E1 and have engineered the resulting cDNAs in plasmids suitable for in vitro transcription/translation and in vivo expression. The results we obtained with this approach show that (i) internal signal sequence(s) are present upstream of E1, and this protein may be generated and inserted in the endoplasmic reticulum membrane independently of the PE2 signal sequence; and (ii) E1 is efficiently transported to the plasma membrane in the absence of PE2/E2; exit from the endoplasmic reticulum of E1 takes place with almost the same timing in the presence or in the absence of PE2.