The envelope of alphavirus particles contains two major glycoproteins, E1 and E2, that participate in virus entry and assembly of new virus particles. Interactions between these glycoproteins determine their correct functioning. The expression of each glycoprotein in the absence of the other counterpart was achieved by means of electroporation of modified Sindbis virus (SV) genomes. In addition, in trans coexpression of both glycoproteins was also tested in BHK cells. Synthesis of the E1 glycoprotein alone gave rise to cell fusion after incubation in low-pH medium. In addition, expression of E1 in the absence of the E2 precursor, PE2 (E3+E2), induced the formation of cytoplasmic vacuoles in the transfected cells. The normal phenotype was recovered when PE2 was coexpressed in trans with E1. Moreover, this coexpression modified the processing of the PE2 glycoprotein. PE2 synthesized in the absence of E1 gave rise to a product, E2', whose migration was slower in SDS-polyacrylamide gel than that of genuine E2 from SV-infected cells. This alteration was corrected upon in trans coexpression of E1 and PE2. These results suggest that the two glycoproteins, E1 and PE2, interact after their expression from two separate SV genomes. Notably, BHK cells cotransfected with the two modified genomes produced SV particles. Our findings suggest that SV E1 and E2 synthesized in trans can interact with each other and participate together with capsid protein in the assembly of new virus particles.