We found serological evidence of infection with Prospect Hill virus, a Hantaan-like virus isolated from meadow voles (Microtus pennsylvanicus), in microtine and cricetid rodents trapped in Maryland, West Virginia, Minnesota and California, USA. Fluorescent antibodies were detected in sera from M. pennsylvanicus (74/277), M. californicus (39/185), Clethrionomys gapperi (5/51), Peromyscus maniculatus (4/22) and P. truei (1/11). Sera from seropositive P. maniculatus contained neutralizing antibodies against Prospect Hill virus, confirming that infection with Prospect Hill virus or antigenically related viruses is not restricted to microtine rodents in the USA. Despite the widespread distribution of Prospect Hill virus in indigenous rodents, the recent demonstration that American mammalogists are only rarely infected supports the view that the overall risk of Prospect Hill virus infection in man is low.