25 skin biopsy isolates of Borrelia burgdorferi sensu lato, mainly from German patients with erythema migrans [EM], borrelial lymphocytoma [BL] and acrodermatitis chronica atrophicans [ACA], were species-differentiated using pulsed-field gel electrophoresis (PFGE). The isolates revealed 15 B. afzelii, 8 B. garinii and 2 B. burgdorferi sensu stricto species according to Miu I digestion. All 6 ACA isolates were identified as B. afzelii species, whereas the 2 borrelial lymphocytoma isolates were grouped into B. garinii species. 12 B. afzelii strains, including 4 ACA isolates, were further investigated by digestion with 5 restriction enzymes. Most of the strains revealed an individual pattern. No specific characteristically large restriction fragment pattern (LRFP) could be detected within the ACA and/or EM group. The ACA isolates could not be clearly differentiated from the EM isolates according to the LRFP patterns. PFGE is thus a highly effective method for detecting differences among B. afzelii isolates.