The detection of antibody to the Borrelia burgdorferi C6 peptide by use of enzyme-linked immunoassays is a widely accepted method for the diagnosis of Lyme disease spirochete infection in dogs and in humans. Antibody to the C6 peptide is highly specific for B. burgdorferi and declines following treatment of dogs and humans exposed to B. burgdorferi. A quantitative assay for determining C6 antibody levels was developed and used to measure changes in antibody levels following antibiotic treatment of B. burgdorferi antibody-positive nonclinical dogs. One hundred thirty-two client-owned dogs were used in the study; 64 were negative, 53 of 68 positive animals received treatment, and 15 were untreated controls. Test sera were collected at 3, 6, and 12 months from seropositive dogs receiving treatment and untreated controls. Dogs in the treated group were assigned to moderate-to-high (> or =29 U/ml)- and low (<29 U/ml)-C6-level groups because the change in the C6 level after treatment was dependent on the level prior to treatment. There were significant declines in the 30 dogs with moderate-to-high initial C6 levels that exceeded the maximal declines of the untreated control dogs in all cases at 6 months (16 data points) and 12 months (29 data points) posttreatment. There was little change in C6 level following antibiotic therapy in the 23 dogs with low initial C6 levels. The quantitative C6 antibody test can be used to measure changes in C6 antibody levels following treatment of antibody-positive nonclinical dogs.