The transmembrane (TM) domains of hepatitis C virus (HCV) envelope glycoproteins E1 and E2 have been shown to play multiple roles during the biogenesis of the E1E2 heterodimer. By using alanine scanning insertion mutagenesis within the TM domains of HCV envelope glycoproteins, we have previously shown that the central regions of these domains as well as the N-terminal part of the TM domain of E1 are involved in heterodimerization. Here, we used a tryptophan replacement scan of these regions to identify individual residues that participate in those interactions. Our mutagenesis study identified at least four residues involved in heterodimerization: Gly 354, Gly 358, Lys 370, and Asp 728. Interestingly, Gly 354 and Gly 358 belong to a GXXXG oligomerization motif. Our tryptophan mutants were also used to generate retrovirus-based, HCV-pseudotyped particles (HCVpp) in order to analyze the effects of these mutations on virus entry. Surprisingly, two mutants consistently displayed higher infectivity compared to that of the wild type. In contrast, HCVpp infectivity was strongly affected for many mutants, despite normal E1E2 heterodimerization and normal levels of incorporation of HCV glycoproteins into HCVpp. The characterization of some of these HCVpp mutants in the recently developed in vitro fusion assay using fluorescent-labeled liposomes indicated that mutations reducing HCVpp infectivity without altering E1E2 heterodimerization affected the fusion properties of HCV envelope glycoproteins. In conclusion, this mutational analysis identified residues involved in E1E2 heterodimerization and revealed that the TM domains of HCV envelope glycoproteins play a major role in the fusion properties of these proteins.