The live-attenuated yellow fever (YF) vaccine virus, strain 17D-204, has long been known to consist of a heterologous population of virions. Gould et al. (J. Gen. Virol. 70, 1889-1894 (1989)) previously demonstrated that variant viruses exhibiting a YF wild-type-specific envelope (E) protein epitope are present at low frequency in the vaccine pool and were able to isolate representative virus variants with and without this epitope, designated 17D(+wt) and 17D(-wt), respectively. These variants were employed here in an investigation of YF virus pathogenesis in the mouse model. Both the 17D-204 parent and the 17D(+wt) variant viruses were lethal for adult outbred mice by the intracerebral route of inoculation. However, the 17D(-wt) variant was significantly attenuated (18% mortality rate) and replicated to much lower titer in the brains of infected mice. A single amino acid substitution in the envelope (E) protein at E-240 (Ala-->Val) was identified as responsible for the restricted replication of the 17D(-wt) variant in vivo. The 17D(+wt) variant has an additional second-site mutation, believed to encode a reversion to the neurovirulence phenotype of the 17D-204 parent virus. The amino acid substitution in the E protein at E-173 (Thr-->Ile) of the 17D(+wt) variant which results in the appearance of the wild-type-specific epitope or nucleotide changes in the 5' and 3' noncoding regions of the virus are proposed as a candidates.