Recent studies have demonstrated the importance of heptad repeat regions within envelope proteins of viruses in mediating conformational changes at various stages of viral infection. However, it is not clear if heptad repeats have a direct role in the actual fusion event. Here we have synthesized, fluorescently labeled and functionally and structurally characterized a wild-type 70 residue peptide (SV-117) composed of both the fusion peptide and the N-terminal heptad repeat of Sendai virus fusion protein, two of its mutants, as well as the fusion peptide and heptad repeat separately. One mutation was introduced in the fusion peptide (G119K) and another in the heptad repeat region (I154K). Similar mutations have been shown to drastically reduce the fusogenic ability of the homologous fusion protein of Newcastle disease virus. We found that only SV-117 was active in inducing lipid mixing of egg phosphatidylcholine/phosphatidyiglycerol (PC/PG) large unilamellar vesicles (LUV), and not the mutants nor the mixture of the fusion peptide and the heptad repeat. Functional characterization revealed that SV-117, and to a lesser extent its two mutants, were potent inhibitors of Sendai virus-mediated hemolysis of red blood cells, while the fusion peptide and SV-150 were negligibly active alone or in a mixture. Hemagglutinin assays revealed that none of the peptides disturb the binding of virions to red blood cells. Further studies revealed that SV-117 and its mutants oligomerize similarly in solution and in membrane, and have similar potency in inducing vesicle aggregation. Circular dichroism and FTIR spectroscopy revealed a higher helical content for SV-117 compared to its mutants in 40 % tifluorethanol and in PC/PG multibilayer membranes, respectively, ATR-FTIR studies indicated that SV-117 lies more parallel with the surface of the membrane than its mutants. These observations suggest a direct role for the N-terminal heptad repeat in assisting the fusion peptide in mediating membrane fusion.