The nucleotide sequence of the glycoprotein genes of fully virulent Sindbis virus and derived mutants that have reduced neurovirulence for neonatal mice (attenuated mutants) has been determined. A single amino acid difference, arginine instead of serine at position 114 of the mature E2 glycoprotein, distinguished the prototype attenuated mutant from its virulent wild-type parent. Virulent revertants of the attenuated mutant showed same-site reversion to the wild-type sequence. An identical single amino acid substitution, an arginine for the serine at E2 position 114, was found in a second independently selected attenuated mutant. The strains are characterized by genetic linkage between attenuation, accelerated penetration of baby hamster kidney cells, and efficient neutralization by the E2-specific monoclonal antibodies R6 and R13; selection for change in one property simultaneously selected for change in the other two (Olmsted, R. A., Baric, R. S., Sawyer, B. A. & Johnston, R. E. (1984) Science 225, 424-427 and Olmsted, R. A., Meyer, W. J. & Johnston, R. E. (1986) Virology 148, 1-10). The nucleotide sequence data suggest that a single mutation in the E2 gene is sufficient to cause these coordinate phenotypic changes. These findings identify a single locus in a Sindbis virus surface glycoprotein gene that determines both efficiency of interaction with cultured baby hamster kidney cells and degree of virulence in neonatal mice.