AbstractHuman serum samples collected from healthy individuals in 14 counties were screened by ELISA in order to investigate the presence of hantavirus infections in Estonia. Out of 1,234 serum samples, 124 were found positive for hantavirus‐specific IgG and were subsequently serotyped by a focus reduction neutralization test. A total of 112 samples neutralized at least one of the examined hantaviruses—Puumala (PUUV), Saaremaa (SAAV), Dobrava (DOBV), Hantaan, and Seoul viruses—and thereby, the focus reduction neutralization test confirmed the overall hantavirus seroprevalence rate in Estonia to be 9.1%. Most of the sera showed a specific reaction (at least 4‐fold higher endpoint titer) of neutralizing antibodies to PUUV (5.1%), while 3.4% showed a SAAV‐ or SAAV/DOBV‐specific reaction. The fact that seven sera (0.6%) could not be serotyped may indicate the presence of an unknown hantavirus serotype. Hantavirus infections were confirmed in 13 of 14 investigated counties, with highly varying seroprevalence rates (1.0–28.4%). The sex ratio was 1.8:1.0 (M:F), and the antibody prevalence peaked in the age group 45–54 years. A total of 513 rodents of seven species trapped in seven counties were examined for the presence of hantavirus antigen, in order to study the distribution of hantavirus natural carriers. Two species, Clethrionomys glareolus and Apodemus agrarius, were found positive for hantaviral antigen in 13.7% and 4.5% of the investigated rodents, respectively. Analyses of viral sequences recovered from infected C. glareolus tissue samples showed that the infecting virus belonged to the PUUV genotype, confirming that PUUV circulates in mainland Estonia. The Estonian PUUV strains were placed in the closest proximity to Russian PUUV strains in phylogenetic trees, suggesting a common evolutionary history. Together with earlier data on SAAV in A. agrarius, the results revealed that two hantaviruses, PUUV and SAAV, are common in Estonia and that the incidence of human infection is high in both cases. J. Med. Virol. 68:589–598, 2002. © 2002 Wiley‐Liss, Inc.