Three rodent species: Clethrionomys glareolus, Apodemus flavicollis and Microtus arvalis from Mazury Lakes District of Poland were examined for antibodies to Borrelia burgdorferi by enzyme-labelled protein G assay (ELGA). C. glareolus had an exceptionally high prevalence of B. burgdorferi antibodies - 58%, but A. flavicollis and M. arvalis also showed significant prevalence of 16.6% and 10.5%, respectively. The ELGA method is highly specific with good reproducibility. Nevertheless, some differences of sensitivity of assessed samples were season dependent. However, high seroprevalence did not coincide with infestation rates of examined rodents by I. ricinus ticks. The results indicated that in Mazury Lakes District, naturally infected rodents play an important role as an animal reservoir host for B. burgdorferi, and these animals may increase the risk of human infections in some habitats used as recreation areas. Also, this study shows that ELGA method based on the affinity of protein G for IgG of wild animals may be widely used to determine the competent zoonotic reservoir of B. burgdorferi.