We determined by real-time PCR the distribution of Mycobacterium ulcerans DNA in the excised lesion of a Buruli ulcer patient. A new lesion developed adjacent to the site of excision in the patient. The excised margin around the primary lesion contained a small amount of mycobacterial DNA in the area where the secondary lesion developed. These results suggest that a relatively small number of infiltrating mycobacteria can lead to the development of a recurrence.