Francisella tularensis subsp. tularensis is the common causal agent of tularemia in the USA and Canada, while F. tularensis subsp. palaearctica (holarctica) occurs in Europe, Asia, and to a minor extent in North America. F. tularensis subsp. mediaasiatica was found only in central Asia in a part of the former Soviet Union. Of the total of 155 F. tularensis strains isolated over the years 1978-1996 during the surveillance of tularemia in Slovakia, 65 were from small mammals, 68 from ticks and 22 from mites and fleas. They were characterized and classified by basic markers of infraspecific taxonomy in tests in vitro and compared with type strains of three subspecies and biovars of F. tularensis. Comparative studies have revealed biological properties characteristic of F. tularensis subsp. tularensis in 17 strains isolated from fleas and mites parasiting on small terrestrial mammals, collected in the Danube region, near Bratislava. These strains fermented glycerol, glucose, were positive for citrulline ureidase and sensitive to erythromycin, in contrast to the other 138 isolates classified as F. tularensis subsp. palaearctica (holarctica), biovar II, which fermented only glucose, were negative for citrulline ureidase and resistant to erythromycin. Two selected pairs of isolates with properties characteristic of F. tularensis subsp. palaearctica (holarctica), biovar II (SE-210, SE-234) and of F. tularensis subsp. tularensis (SE-219, SE-221), as shown in tests in vitro, were further examined for their pathogenicity on white mice, guinea pigs and domestic rabbits. In tests of virulence on domestic rabbits, the isolates SE-210 and SE-234 had low pathogenicity, while the isolates SE-219 and SE-221 exhibited high pathogenicity, which along with their biochemical properties confirmed their identification as strains of F. tularensis subsp. tularensis. The first findings of the highly virulent strains of F. tularensis subsp. tularensis in Europe indicate a serious event from epidemiologic and epiozootologic aspects, requiring systematic surveillance.