Rio Grande (RG) virus, a new member of the Phlebotomus fever serogroup, was inoculated into wild wood rats (Neotoma micropus) and laboratory-reared cotton rats (Sigmodon hispidus) to determine if these potential hosts could be experimentally infected. Nine of 14 (64%) wood rats became viremic, with titers of circulating virus ranging from 10(2.3) to 10(5.3) plaque-forming units (PFU)/ml and a geometric mean titer of 10(3.7) PFU/ml. Virus was not detected in urine specimens from inoculated wood rats but was found in a single saliva specimen. RG virus was detected in the blood of 1 of 12 (8%) cotton rats. Neutralizing (N) antibody developed in 8 of 9 inoculated wood rats which survived for 30 days postinoculation and in 11 of 12 cotton rats. N antibody was still detectable in 4 of 7 wood rats which survived for 1 year, and all 7 were resistant to rechallenge with the virus, as were 3 wood rats with naturally-acquired antibody. High mortality (36%) occurred in inoculated wood rats; whereas low mortality (8%) occurred in cotton rats. The specific cause of death of the rats was not determined. Modes of transmission of the virus in nature are discussed.