Four viable West Nile (WN) 3'SL-mutant viruses were evaluated for neuroinvasiveness and neurovirulence in mice. All mutants were highly attenuated for neuroinvasiveness. However, only one of these four (WNmutE virus) was significantly attenuated for neurovirulence. To attenuate WNmutE virus further, we introduced five substitution mutations into the envelope (env) gene segment in wild-type (wt) WN and WNmutE genomes, based on differences in the env gene sequence between the live Japanese encephalitis vaccine (SA14-14-2) and its virulent parent. The env gene mutations had an attenuating effect in the context of the wt WNV genome but only a marginal enhancing effect on the attenuation of WNmutE virus.