The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20-30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.