Infestation of small mammals, including common shrews Sorex araneus L., field mice Apodemus agrarius Pallas, and red voles Clethrionomus rutilus Schreber, with immature Ixodes persulcatus ticks and their infection with tickborne encephalitis virus (TBEV) were studied in the forest-steppe habitat in the vicinity of Novosibirsk, Russia. Larval ticks parasitize all three host species, but virtually all nymphs were found only on field mice and red voles. Detection of the viral RNA using reverse transcription (RT) with subsequent nested polymerase chain reaction (nPCR) and of viral antigen using enzyme-linked immunosorbent assay (ELISA) revealed a high prevalence of TBEV-positive animals in both the summer and winter. The proportion of small mammals with hemagglutination inhibition (HI) antibodies was significantly lower than with ELISA-detected antibodies. Taken together, the data suggest that small mammals may maintain TBEV as a persistent infection throughout the year.