Serum samples from 100 horses in Flanders with no clinical evidence of Lyme disease were examined for the presence of IgG antibodies against Borrelia burgdorferi sensu lato using an indirect ELISA. In 36 of the samples antibodies against this bacterium were detected, indicating that infections with B. burgdorferi sl regularly occur in horses in Flanders. These findings also indicate the potential exposure of humans to this spirochete in this region.