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To determine whether the tick-borne encephalitis virus (TBEV) is present in vector ticks and mammalian hosts in Korea, we examined two tick species, Haemaphysalis longicornis (n = 548) and Ixodes nipponensis (n = 87), and the lungs or spleens of rodents Apodemus agrarius (n = 24) and wild boars (n = 16). Tick-borne encephalitis virus was detected in samples by reverse transcriptase (RT)-nested polymerase chain reaction (PCR), after which TBEV-positive samples were inoculated into BHK-21 cells and suckling mice. Tick-borne encephalitis virus genes were detected in 4 of 38 tick pools and 5 of 24 wild rodents. Suckling mice inoculated intracerebrally with TBEV-positive rodent samples showed signs of encephalitis at six days post-inoculation. The isolation of TBEV was confirmed by inoculating samples obtained from the brains of sick mice in cell culture. Phylogenetic analysis showed that the E genes of the TBEV isolates were clustered with the Western subtype (98% identity). This study suggests the possible occurrence of tick-borne encephalitis in Korea.