Although it is known that two-tier serologic testing for Lyme disease may be associated with false positive results on the IgM immunoblot, this problem has never been systematically studied in the clinical practice setting. In a retrospective investigation of patients referred to the private adult practice of an Infectious Diseases physician for possible for Lyme disease, 50 of 182 patients (27.5%, 95% CI: 21.1-34.6) were found to have a false positive IgM immunoblot. 78.0% of these patients had received unnecessary antibiotic therapy. False positive results were not restricted to any single commercial laboratory. Research on alternative testing strategies that eliminate the IgM immunoblot entirely is warranted.