The frequency and distribution of La Crosse (LAC) virus overwintering in Aedes triseriatus diapause eggs were studied during 1974 by following 64 tree-hole oviposition sites in four enzootic hardwood forest areas. A direct fluorescent antibody technique, adapted for detection of LAC virus in individual mosquitoes, proved to be a rapid, reliable and economical tool for ascertaining true field infection rates. Virus was found in larvae from each of the four study areas before the seasonal emergence of adults and detected in 10 (0.6%) of 1,698 individually processed adults reared from collected larvae. In one of these study areas, all 12 located tree-holes were enclosed with screen before the seasonal emergence of adults, to ensure that all collected from these sites originated from overwintered eggs. Of 1,280 Aedes triseriatus processed from this area throughout the season, 16 (1.2%) contained virus. Isolates were found in overwintered Aedes triseriatus throughout the summer months, demonstrating the role of these tree-hole sites as foci for both overwintering and continuing summer season source of LAC virus in these forested areas.