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Two tick-borne encephalitis (TBE) virus variants were studied: mouse brain-adapted strain EK-328 and its derivate adapted to Hyalomma marginatum ticks. The tick-adapted virus exhibited small-plaque phenotype and slower replication in PEK cells, higher yield in ticks, decreased neuroinvasiveness in mice, increased binding to heparin-sepharose. A total of 15 nucleotide substitutions distinguished genomes of these variants, six substitutions resulted in protein sequence alterations, and two were in 5'NTR. Two amino acid substitutions in E protein were responsible for the observed phenotypic differences. Data obtained during reverse passaging of the tick-adapted virus in vivo and in vitro suggest that TBE virus exists as a heterogeneous population that contains virus variants most adapted to reproduction in either ticks or mammals. Host switch results in a change in the ratio of these variants in the population. Plaque purification of the tick-adapted virus resulted in the prompt emergence of new mutants with different virulence for mammals.