Persistence of Borrelia burgdorferi DNA in tissues following antibiotic treatment was evaluated in C3H mice inoculated intradermally with 10(3) B. burgdorferi N40 or sterile medium. Half of the infected mice and all of the uninfected mice were treated with ceftriaxone 15 days after inoculation for 5 days. Ear and urinary bladder samples were collected on days 20, 30, and 60 after inoculation for culture and for extraction of DNA and amplification of specific spirochetal DNA by polymerase chain reaction (PCR). PCR primers were specific for a 280-bp portion of a highly conserved region of the gene encoding outer surface protein (Osp) A of B. burgdorferi and for a 328-bp part of the OspB gene. There was excellent concordance between culture and PCR for ears (35/36 mice) and bladders (33/36). Both tissues became uniformly negative at the earliest interval tested after antibiotic treatment. Thus, the ability to amplify B. burgdorferi DNA quickly disappeared from tissues that had become culture-negative after antibiotic treatment, suggesting that serial study of PCR-positive tissues and fluids may be useful for evaluating the efficacy of antibiotic therapy in human Lyme disease.