Potosi (POT) virus (Bunyaviridae) was isolated from Aedes albopictus, an introduced Asian mosquito species, collected at a used tire yard in Potosi, Missouri (USA), in August and September, 1989. In September, 1990, small animals were trapped at the tire yard and six cattle were sampled at an adjacent farm; in November 1990 and 1991, blood samples were collected with filter paper strips from 364 hunter-killed, white-tailed deer (Odocoileus virginianus) in the region to determine the possible reservoir hosts of the virus. Deer specimens from Arkansas (n = 70), Colorado (n = 29), and Iowa (n = 763) (USA) were also analyzed. Specimens from 33 small vertebrates captured at the tire yard were negative for viruses. Only one eastern chipmunk (Tamias striatus) and none of six cattle had neutralizing (N) antibody against POT virus by the plaque-reduction serum neutralization test in Vero cell culture but 45 (25%) of 178 deer specimens in 1990 and 55 (30%) of 186 in 1991 were antibody positive. The 186 deer sera from 1991 were tested further and 29 (16%) were also N antibody positive to Cache Valley (CV) virus. From the 763 deer specimens tested from Iowa in 1993, 114 (15%) had N antibody to POT virus. Of 70 serum specimens from Arkansas deer in 1990, 33 (47%) had N antibody to POT and 15 (21%) to CV viruses; two (7%) of 29 CV negative serum specimens from Colorado deer in 1981 were serologically positive to POT virus. Three eastern chipmunks were experimentally inoculated with POT virus to determine their reservoir potential; none became viremic but all developed N antibody. Thus we propose that POT virus may be another virus regularly infecting wild deer populations but its impact on the health of these animals is unknown.