Erythema migrans skin lesions resulting from a tick bite and infection with Borrelia burgdorferi sensu lato eventually resolve, even without antibiotic therapy. The aim of the present study was to gauge the frequency of persistence of B. burgdorferi sensu lato in such lesions. Thus, the site of a previous lesion was biopsied and cultured in 48 patients: 39 with systemic or localized symptoms/signs and nine with no symptoms. B. burgdorferi sensu lato was isolated from biopsied skin from three symptomatic patients. Cultures of other tissues and fluids were negative. By genospecies-specific polymerase chain reaction (PCR) and pulsed-field gel electrophoretic (PFGE) analysis, two isolates were classified as Borrelia afzelii. The remaining isolate was a member of an unusual group of strains that type as B. burgdorferi sensu stricto by genospecies PCR but possess an atypical PFGE profile. All three patients had a dramatic clinical response to antibiotic treatment. These findings demonstrate the capacity of viable B. burgdorferi sensu lato organisms to persist in clinically normal-appearing skin at the site of a resolved erythema migrans rash for periods ranging from 2 months to 3.5 years. This observation may provide new insight into the organisms' ability to evade the host's immune response.