It has been suggested that a <4-fold decline in the immunoglobulin G (IgG) antibody response to the VlsE sixth invariant region peptide of Borrelia burgdorferi within 6 months after antibiotic treatment may indicate spirochetal persistence in Lyme disease. We studied the response to this peptide in 77 patients with early or late disease, for whom archival samples were available at the time of antibiotic treatment and approximately 6 months or years later. Eight (33%) of the 24 patients with early manifestations and 18 (86%) of the 21 patients with late manifestations had a <4-fold decline in IgG anti-VlsE titers approximately 6 months after successful antibiotic treatment. Of 32 additional patients, 13 (50%) with early manifestations and 5 (83%) with late manifestations still had positive anti-VlsE titers 8-15 years after successful antibiotic treatment. We conclude that persistence of the anti-VlsE antibody response for months or years after antibiotic treatment cannot be equated with spirochetal persistence in Lyme disease.