In 2002, two cases of tick-borne encephalitis (TBE) were diagnosed in inhabitants of a tick endemic area on the island of Aspö in southeastern Sweden. During the previous 25 y, only two other cases of TBE had been diagnosed in that region of Sweden. To investigate the presence and evolution of seroprevalence of antibodies to TBE virus (TBEV), we compared inhabitants´ anti-TBEV immunoglobulin G (IgG) levels in blood samples drawn in 1991 and 2002. A significant increase in IgG antibody levels was observed in 24/200 (12.0%) 2002 blood samples compared to 7/200 (3.5%) 1991 samples. However, neutralizing antibodies were detected in only 4 of the two-step enzyme-linked immunosorbent assay (ELISA) IgG positive sera against TBEV, corresponding to a TBEV neutralizing test (NT) prevalence of 2%. Significantly more men than women were seropositive for TBEV antibodies. Compared to other age groups, the greatest increase in TBEV antibody levels was observed in the 20-29 y age group. However, the majority of seropositive samples were from participants >50 y of age. Recommending preventative measures, including vaccination against TBE, to individuals who reside in or regularly visit TBEV endemic areas is suggested.