Skin biopsy specimens from the peripheral aspect of erythema migrans lesions (site 1) and from clinically normal perilesional areas (site 2) were compared as sources of Borrelia burgdorferi. This spirochete was isolated from the skin of 18 of 21 (86%) patients with untreated early Lyme disease at one or both biopsy sites. Site 1 specimens were superior to site 2 specimens for the isolation of B. burgdorferi. Site 1 specimens from 18 (86%) patients were culture positive, and site 2 specimens from 12 (57%) patients were culture positive. For patients whose site 2 specimens were culture positive, site 1 specimens were also found to be culture positive. B. burgdorferi was isolated from two patients with atypical lesions and from two patients with erythema migrans lesions that were less than 5 cm in diameter. This study demonstrates that the cultivation of B. burgdorferi from skin biopsy specimens from cutaneous lesions thought to be erythema migrans can be an efficacious procedure for confirming the diagnosis of Lyme disease and that the spirochete is present in clinically normal appearing perilesional skin.