A blinded cohort study was conducted in 2000 to better understand the emergence of La Crosse virus infection in eastern Tennessee, with special emphasis on the potential mosquito vector(s). Children with suspected central nervous system infection were enrolled at the time of clinical presentation at a large pediatric referral hospital. Clinical, environmental, and entomological data were collected prior to case confirmation. Sixteen of the 40 children included in the final analysis were confirmed to have La Crosse infection by a fourfold increase in antibody titers between collection of acute- and convalescent-phase sera. Factors significantly associated with La Crosse infection included average number of hours per day spent outdoors (5.9 for La Crosse virus cases vs. 4.0 for noncases, p = 0.049); living in a residence with one or more tree holes within 100 m (relative risk = 3.96 vs. no tree holes within 100 m, p = 0.028); and total burden of Aedes albopictus (number of female and male larvae and adults collected at a site), which was more than three times greater around the residences of La Crosse virus cases versus noncases (p = 0.013). Evidence is accumulating that the newly introduced mosquito species Ae. albopictus may be involved in the emergence of La Crosse virus infection in eastern Tennessee.