Eight enzootic strains of western equine encephalomyelitis (WEE) virus isolated from Culex tarsalis or Aedes melanimon collected in several geographic areas of California were evaluated for their virulence in suckling mice, adult mice, and one-day-old baby chickens. The epidemic Fleming strain and the cloned B628(Cl 15) variant were used as virulent and avirulent control viruses, respectively, in adult mice. Enzootic strains of WEE virus were grouped into three phenotypes on the basis of their neurovirulent and neuroinvasive properties in adult mice. Three strains possessed high neurovirulence and were neuroinvasive; three strains had intermediate neurovirulence and lacked neuroinvasiveness; and two strains had low to nil neurovirulence and were non-neuroinvasive. In fact, five of the eight enzootic strains lacked neuroinvasiveness. Interestingly, highly virulent enzootic strains of WEE virus were isolated from Cx. tarsalis collected in the Sacramento Valley during 1994 and 1995 in the absence of identified human disease. The Fleming strain, the B628(Cl 15) variant, and four enzootic strains from the Sacramento Valley were virulent for baby chickens following subcutaneous inoculation. Thus, inoculation into baby chicks cannot discriminate between WEE viruses that are virulent and avirulent for adult mice.