In an area where Lyme disease is endemic in The Netherlands all dogs had positive titers by whole-cell enzyme-linked immunosorbent assay and appeared to be naturally infected by Borrelia burgdorferi sensu lato. To compare the antibody responses of symptomatic dogs and asymptomatic controls, we performed Western blots and in vitro immobilization assays to study antibody-dependent bactericidal activity. Strains from three different genospecies were employed as the antigen source: B. burgdorferi strain B31, Borrelia garinii strain A87S, and Borrelia afzelii strain pKo. Antibodies against flagellin (p41) and p39 for three strains were found in sera from both symptomatic and asymptomatic dogs and were therefore considered to be markers of exposure. Antibodies against p56 and p30 of strain B31, against p75, p58, p50, OspC, and p<19 of strain A87S, and against p56, p54, p45, OspB, p31, p26, and p<19 of strain pKo were found significantly more frequently in sera from symptomatic dogs younger than 8 years when the first symptoms were observed than in those from age-matched controls (P < 0.01). These antibodies were not found in preclinical sera and appeared during development of disease. Antibodies against OspA of strains B31 and A87S were only seen in acute-phase and convalescent sera from three dogs that recovered from disease. Incubation with 25% normal canine serum did not result in the immobilization of strains B31 and pKo, but partial immobilization of strain A87S (61% ± 24% [standard deviation] at 5 h) occurred. Seven of 15 sera from symptomatic dogs but none of the sera from 11 asymptomatic dogs had antibody-dependent immobilizing activity against one of the strains. Consecutive sera from one of these dogs immobilized two different strains. Antibody-mediated bactericidal serum was not seen before onset of disease, was strongest in the acute phase of disease, and fluctuated during chronic disease. From seven out of eight symptomatic dogs Borrelia DNA was amplified by PCR; in three of them the bactericidal activity was directed against one of the genospecies amplified from that dog; however, four PCR-positive dogs lacked bactericidal activity. In conclusion, dogs with symptomatic canine borreliosis have more-extensive antibody reactivity against Borrelia, as shown by both Western blotting and immobilization assays.