The prevalence of antibodies to Neospora caninum was examined in European bison (Bison bonasus bonasus L.) living in free and fenced areas in Poland. Sera of 320 European bison, different ages and sexes, from breeding areas in Poland were tested for N. caninum antibodies using ELISA test. Positive antibody responses were found in 23 bison (prevalence 7.3%). Additionally, all positive sera were tested by Western blot to verify the ELISA results. The Western blot results confirmed the presence of antibodies to Neospora tachyzoites antigens in all 23 sera tested. The antibodies were detected against a wide range of NC-1 tachyzoite antigens. The antibody responses were directed against proteins at: 9.5, 17, 21, 27, 31, 36.5, 38, 40, 43, 47, 48.5, 53.5 and 58 kDa. The most heavily stained bands had molecular weights of 9.5, 17, 27 and 58 kDa. The most important is that antibody to N. caninum was detected for the first time in sera from bison cow shot in 1988. It is the year of recognition of this protozoan parasite. Our results indicate strongly the presence of N. caninum in European bison in Poland and suggest that a sylvatic cycle of N. caninum can exist. However, further studies are needed to evaluate the existence of a sylvatic cycle of N. caninum. The study on the effect of the infection on the health status and conservation of European bison should be taken under consideration too.