La Crosse (LAC) virus was isolated from the blood of seven chipmunks (Tamias striatus) captured during the summer of 1970 in southwestern Wisconsin. With the exception of the original isolate obtained from human brain after fatal encephalitis, these represent the first known isolations of LAC virus from a naturally infected free-living vertebrate. The chipmunks were trapped and periodically recaptured in two study areas where 59 chipmunks became infected and developed neutralizing antibody during the summer cycle of virus transmission. All isolates were obtained from blood samples collected within a 7 week period between 11 July and 23 August, and all were from seronegative chipmunks; 6 of these were recaptured and found to be seropositive 2 to 3 weeks later. The isolates proved identical to each other in comparative micro-neutralization tests using BHK21 cell cultures and immune chipmunk serum or hyperimmune mouse ascitic fluids. Neutralization tests showed the isolates to be different from snowshoe hare, trivittatus, and Jamestown Canyon prototype virus strains but indistinguishable from the LAC prototype. Findings demonstrate multiplication and transmissibility of LAC virus in a naturally infected host and are consistent with the thesis that chipmunks are important amplifying hosts for LAC virus and that Aedes triseriatus mosquitoes serve as vectors in transmitting their infections. Ecological significance of the findings are discussed in regard to current perspectives.