We conducted a small mammal trapping study to investigate temporal variation in prevalence of infection in hantavirus reservoir populations in the Patagonian Andes mountain range, Rio Negro province, Argentina. Rodent blood samples collected in natural and periurban habitats and at the home of an hantavirus pulmonary syndrome (HPS) case patient were analysed by enzyme-linked immunosorbent assay. Organ tissue samples were tested by polymerase chain reaction (PCR) and nucleotide sequence analysis. Eight species of 1032 rodents were captured in 15 551 trap nights, giving an overall trap success of 6.6%. Hantavirus antibody was detected in 30 of 555 Oligoryzomys longicaudatus (reservoir of Andes virus), three of 411 Abrothrix longipilis, and one of 10 Loxodontomys micropus. Antibody prevalences in O. longicaudatus were 13.7% in spring 1996, 59.3% in summer 1996, 2.1% in autumn 1997, 12.4% in winter 1997 and 3.1% in spring 1997. A much higher antibody prevalence (33%) was found during trapping around the residence of an HPS case patient. Higher prevalences were found in older male O. longicaudatus. There was no apparent correlation of antibody prevalence with rodent population density, or of rodent population density or antibody prevalence with numbers of human cases. For an HPS case that occurred in our study area in 1997, we identified the probable rodent reservoir and likely site of exposure by matching the genetic sequences of virus obtained from a rodent and the HPS case patient.