In the course of epizootological research on Lyme borreliosis in domestic and farm animals, the serological evidence for the occurrence of this zoonosis in cattle was screened. An ELISA showed that 25.2% of cattle from seven geographical areas in Slovakia were positive for anti-Borrelia IgG antibodies. In particular localities, the seroprevalence ranged from 0.6% to 34.3%. Of 33 cases with clinical signs, 20 ELISA-positive samples were also confirmed in Western blots. The most frequent clinical signs were lameness and swollen joints, but most of the cases were asymptomatic. The occurrence of Borrelia burgdorferi antibodies and suspected clinical signs in cattle of Slovak regions indicates that veterinarians should pay attention to this disease in their clinical practice and include it within the differential diagnosis.