rio negro virus (rnv) (venezuelan equine encephalitis subtype vi) circulates only in argentina; in northern provinces, isolates have been obtained from mosquitoes and rodents since 1980 and have been associated with acute febrile illness in humans. however, no studies of rnv have been performed in the central area of the country. we carried out molecular and serological detection of rnv in córdoba, a province of the central part of the country, in mosquitoes and humans, respectively. one mosquito pool tested positive for alphavirus rna by reverse transcriptase-nested polymerase chain reaction (rt-nested pcr). subsequent sequencing determined that this alphavirus grouped with rnv. serological studies detected antibodies to rnv in one human serum sample, which was obtained during the same period that rnv was detected using the aforementioned molecular methods. this is the first report of rnv circulation in the central area of argentina, indicating an expansion of its original distribution. these results highlight the importance of strengthening surveillance procedures in endemic areas, as well as in new regions where rnv may emerge.