The fusion of lipid enveloped viruses with cellular membranes is thought to be mediated by the insertion into the target membrane of the N-terminal polypeptides of viral spike glycoproteins. Since membrane destabilization is a necessary step in membrane fusion, we investigated whether synthetic peptides with amino acid sequences corresponding to the N-termini of influenza virus hemagglutinin (HA2), vesicular stomatitis virus G-protein and Sendai virus F-protein, induce the destabilization and fusion of phospholipid vesicles. Membrane destabilization by the peptides was monitored by the release of aqueous contents of large unilamellar phospholipid vesicles. Aggregation was detected by a resonance energy transfer assay. Membrane fusion was followed by means of assays for the intermixing of phospholipids and of aqueous contents. The 17-amino acid HA2 peptide (HA2.17) destabilized phosphatidylcholine (PC) vesicles even at neutral pH, but the rate and extent of destabilization increased at lower pH. This peptide did not mediate appreciable release of contents from phosphatidylserine (PS) vesicles. HA2.17 induced neither aggregation nor fusion of PC or PS vesicles. In contrast, the 7-amino acid N-terminal peptide of G-protein (G.7) destabilized PS-containing membranes and not pure PC vesicles. Although G.7 caused aggregation of and lipid mixing between PS vesicles, it did not mediate any detectable intermixing of aqueous contents. The presence of cholesterol in PC membranes did not affect the destabilization caused by the N-terminal peptide of Sendai virus F-protein (F1.7), suggesting that cholesterol is not necessary for the effective interaction of this peptide with membranes, contrary to earlier proposals. Our results support the hypothesis that the hydrophobic N-terminal region of certain viral envelope proteins insert into and destabilize target membranes.