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The roles of various subtypes of the California serogroup viruses as infectious agents and as neuropathogens were evaluated by using the plaque reduction neutralization test. Sera from 394 patients with central nervous system (CNS) infections during 1971–1982 and from 501 persons without CNS manifestations were studied. Jamestown Canyon (JC) and La Crosse (LAC) viruses were found to have been common infectious agents in New York State for at least 16 years. JC virus was the prevalent indicated agent in patients with antibody to California serogroup viruses in screening tests (62 of 93 cases), followed by LAC virus (11 cases), snowshoe hare (2 cases), and trivittatus (1 case). In the remaining 17 patients the subtype was undetermined. LAC virus appears to be more pathogenic for children and to produce more serious illness, as judged by the frequent clinical diagnosis of encephalitis. JC virus affects mainly adults, and meningitis was the most common diagnosis. JC virus appears to cause a stronger neutralizing antibody response than does LAC virus, with a longer persistence of high levels of antibody. Some cases of JC virus infection may have been missed in the past due to the choice of a LAC-like isolate from New York State as the sole antigen in hemagglutination-inhibition (HI) screening tests. Comparison of the HI test and a single-dilution neutralization assay for screening for the two major subtypes, JC and LAC, indicated that the latter procedure is more broadly reactive and is less likely to miss cases if only one test antigen is used.