Semliki Forest virus (SFV), an alphavirus, infects cells via a low pH-triggered membrane fusion reaction that takes place within the cellular endocytic pathway. Fusion is mediated by the heterotrimeric virus spike protein, which undergoes conformational changes upon exposure to low pH. The SFV E1 spike subunit contains a hydrophobic domain of 23 amino acids that is highly conserved among alphaviruses. This region is also homologous to a domain of the rotavirus outer capsid protein VP4. Mutagenesis of an SFV spike protein cDNA was used to evaluate the role of the E1 domain in membrane fusion. Mutant spike proteins were expressed in COS cells and assayed for cell-cell fusion activity. Four mutant phenotypes were identified: (i) substitution of Gln for Lys-79 or Leu for Met-88 had no effect on spike protein fusion activity; (ii) substitution of Ala for Asp-75, Ala for Gly-83, or Ala for Gly-91 shifted the pH threshold of fusion to a more acidic range; (iii) mutation of Pro-86 to Asp, Gly-91 to Pro, or deletion of amino acids 83 to 92 resulted in retention of the E1 subunit within the endoplasmic reticulum; and (iv) substitution of Asp for Gly-91 completely blocked cell-cell fusion activity without affecting spike protein assembly or transport. These results argue that the conserved hydrophobic domain of SFV E1 is closely involved in membrane fusion and suggest that the homologous region in rotavirus VP4 may be involved in the entry pathway of this nonenveloped virus.