Summary: Three closely related and probably identical viruses were isolated from mosquitoes (Aedes dorsalis and Culex tarsalis) from Kern County, California. These are believed to be 3 strains of a single virus not previously described or identified. Various characteristics of this agent, named California encephalitis virus, were studied and the following was learned: In certain laboratory animals all three strains produced encephalitis that differs neither clinically nor pathologically from that caused by the other arthropodborne encephalitis viruses in the same area (St. Louis and western equine). Encephalitis, sometimes fatal, developed in mice, cotton rats and hamsters, particularly after intracerebral inoculation. Virus titers of an LD50 level of 10-5 to 10-6 were attained in the brains of infected mice. Guinea pigs, ground squirrels, rabbits, a calf and a monkey, all made a serologic response, but there was no clinical evidence of infection. The squirrel and rabbit are of particular epidemiologic interest, as viremia, necessary for vector infection, was demonstrated. Chickens were completely refractory to infection. Growth of the virus was supported well in the embryo and other tissues of the hen's egg and virus titers were as high as 10-4. In the neutralization and/or vaccination-challenge tests, this virus was not related to western equine, St. Louis, Japanese B, West Nile, yellow fever, Rift Valley fever, Colorado tick fever, Anopheles A or B, Wyeomyia, Bwamba fever, Ilhéus, Theiler's TO or GDVII, herpes simplex or vesicular stomatitis (New Jersey and Indiana strain) viruses. There was a definite crossing shown by cross neutralization and vaccination challenge tests with a virus isolated from Aedes trivitattus from North Dakota, indicating a very close relationship but probable lack of complete identity. With complement-fixing antigen prepared from infected mouse brains, there was no discernible cross reaction with eastern equine or western equine viruses. Undiluted California virus antigen did react in low titer against St. Louis and Japanese B immune sera, but there was no cross reaction when undiluted antigens of these two viruses were tested against California virus antiserum. This virus is roughly 60 to 125 mμ in diameter. Present information indicates this agent probably gives rise to inapparent infections in the wild mammals of the vicinity and certain mosquitoes in the area pick it up from them and pass it along to other hosts, including man and horse. Evidence presented in the first paper of this series indicates that clinical encephalitis may be produced occasionally in man. On the basis of all the available information, it is suggested that the California encephalitis virus be tentatively placed in the group of arthropod-borne encephalitis viruses.