To compare peripheral and central biopsy sites for the isolation of Borrelia burgdorferi sensu lato from typical erythema migrans lesions, two biopsy specimens (one from the margin and the other from the center of the lesion) were taken from each of 53 adult patients. Thirty-four (32.1%) of the 106 biopsy specimens and 23 (43.4%) of the 53 patients were culture-positive. Spirochetes were isolated in 19 (35.9%) of the 53 central and 15 (28.3%) of the 53 peripheral biopsies (nonsignificant difference). In 8 cases only the central specimen, in 4 cases only the peripheral specimen, and in 11 cases both specimens were positive. In a comparison of 36 annular and 17 homogeneous lesions, borreliae were isolated from 15 (41.7%) of the former and 8 (47.1%) of the latter. In the annular-lesion biopsies, 11 central and 12 peripheral specimens were culture-positive. In the homogeneous-lesion biopsies, eight central and three peripheral specimens were culture-positive (nonsignificant difference); there were no culture-positive results solely for the margin of homogeneous lesions. This study demonstrates that B. burgdorferi sensu lato may be isolated from the central aspect of erythema migrans lesions as efficiently as from the peripheral aspect.