We examined the kinetics of membrane fusion of wild type (wt) and Delta6K mutant Semliki Forest virus in a liposomal model system. The final extent of membrane fusion of the mutant (at pH 5.5) was approximately one third that of the wt virus, although the level of E1 (fusion protein) trimerization was, in fact, greater than that of the wt. Studies on the effect of exposure of the viruses to low pH revealed that the Delta6K mutant was inactivated much more rapidly than the wt virus. It is this instability of the mutant particles which probably accounts for the lower fusion levels. Moreover, fusion of the Delta6K mutant was significantly increased by the inclusion of lipid-conjugated heparin in the target liposomes. We conclude that the presence of the 6K protein either in the particle or during the assembly process is important for the correct assembly of the fully infectious SFV particle.