This paper reports the first isolation of Jamestown Canyon (JC) virus from coastal California and the results of tests for antibody to JC virus in mammals living in coastal California. The virus isolation was made from a pool of 50 Aedes dorsalis females collected as adults from Morro Bay, San Luis Obispo County, California. The virus isolate was identified by two-way plaque reduction-serum dilution neutralization tests done in Vero cell cultures. Sera from the mammals were tested for antibody to JC virus by a plaque-reduction serum dilution neutralization method. A high prevalence of JC virus-specific antibody was found in horses and cattle sampled from Morro Bay. This finding is additional evidence for the presence of a virus antigenically identical or closely related to JC virus in Morro Bay and indicates that the vectors of the virus in Morro Bay feed on large mammals. A high prevalence of virus-specific antibody was also found in horses sampled from Marin and San Diego counties. This finding suggests that viruses antigenically identical or closely related to JC virus are geographically widespread in coastal California.