Procedures for the cultural isolation and identification of Borrelia burgdorferi from skin biopsy specimens are described. B. burgdorferi was isolated from 24 of 34 skin biopsy specimens from patients with erythema migrans. Eight of the culture-positive patients had single, primary lesions and 16 had multiple, secondary lesions. The 17 male and 7 female patients were 2 to 70 years old. Biopsy samples were obtained from erythematous or normal-appearing skin within 1 cm of the peripheral aspect of the lesion. Twenty-three of the isolates were detected within 8 days of incubation in Barbour-Stoenner-Kelly medium with no antimicrobial agents. The identities of the isolates were determined by reactivity with monoclonal antibodies H9724 and H5332. Cultivation of B. burgdorferi from skin lesions suggestive of erythema migrans is a practical and clinically relevant procedure. Clinical isolates and corresponding patient sera and urine will contribute to efforts to improve existing immunoserologic testing methods and develop new assays to diagnose Lyme borreliosis.