Sera from 200 randomly selected individuals living in Karksi Nuia, south Estonia, near an area endemic for tick-borne encephalitis and Lyme borreliosis (LB), were tested for antibodies to Borrelia burgdorferi. Antibodies were detected by enzyme-linked immunosorbent assay in 6 individuals (3%; 95% CI: 1-5%), who were middle-aged, asymptomatic anti-nuclear and anti-smooth muscle antibody negative. Our data show that there is low seroprevalence rate of antibodies to B. burgdorferi in an unselected south Estonian population.