The envelope of bovine viral diarrhea virus (BVDV) contains the glycoproteins E rns , E1 and E2. Complementation of a recombinant vesicular stomatitis virus (VSV) with BVDV glycoproteins resulted in infectious pseudotyped viruses. To elucidate the specific role of each of the single envelope glycoproteins during viral entry, pseudotypes were generated bearing the BVDV envelope proteins in different combinations. Pseudoviruses that contained E1 and E2 but not E rns were infectious, indicating that E rns is dispensable for virus entry. VSV/BVDV pseudotypes with chimeric proteins (the ectodomain of the BVDV glycoprotein and the transmembrane domain of the VSV-G protein) were not infectious. The fact that E1-E2 heterodimers were not detected if one of the proteins was chimeric indicated that the heterodimers are crucial for BVDV entry. It was shown by site-directed mutagenesis that the charged amino acids in the transmembrane domains of BVDV E1 (lysine and arginine) and the charged amino acid in the transmembrane domain of E2 (arginine) play a key role in heterodimer formation. Pseudoviruses bearing the mutation E2-R/A, where the charged amino acid was substituted by alanine, were not infectious, supporting the hypothesis that E1-E2 heterodimers are essential for BVDV entry.