Fourteen single and two double point mutants in the highly conserved region (positions 56 to 159) of the G gene of viral hemorrhagic septicaemia virus (VHSV), a salmonid rhabdovirus, were selected and obtained in plasmids by site-directed mutagenesis. Fish cell monolayers transfected with the mutant plasmids were then assayed for protein G (pG) expression, conformation-dependent monoclonal antibody (MAb) reactivity, and cell-cell fusion. Some mutations located in the phospholipid-binding p2 peptide (positions 82 to 110; mutants P86A, A96E, G98A, and R107A) abolished both MAb recognition and fusion activity, while others (P79A, L85S, and R103A) abolished MAb recognition but retained fusion at similar or lower pHs compared to those for the wild type. Phospholipid-binding assays of p2-derived synthetic peptides suggested that phosphatidylserine binding was not affected by the mutations studied. On the other hand, three (P79A, L85S, and T135E) of the four mutants retaining fusion activity mapped around two locations showing amino acid variation in 22 VHSV isolates and in neutralizing MAb-resistant mutants described previously. Mutations located in the hypothetical fusion peptide (positions 142 to 159; mutants F147K, P148K, and W154K) abolished both MAb recognition and fusion activity. The existence of mutants with altered conformation and defective fusion in both p2 and fusion peptides provides further evidence in favor of the participation of these and adjacent regions in some of the steps of the VHSV fusion processes, as suggested by previous studies. In addition, because the studied region induced strong immunological responses in trout, some of the mutants described here might be used to design attenuated VHSV vaccines.