In Europe, at least three species of Borrelia are known to be causative agents of Lyme borreliosis: B. burgdorferi sensu stricto, B. garinii, and B. afzelii. Observable differences in the molecular characteristics of the three species have led to speculation that they may also differ in their pathogenic potential and/or tissue tropisms. Several studies have found an association between the chronic skin manifestation of Lyme borreliosis, acrodermatitis chronica atrophicans, and infection by B. afzelii. We sought to find further evidence for such a correlation by studying the genetic profiles of 22 strains of B. burgdorferi sensu lato derived from 21 patients who presented to the University Medical Center, Ljubljana, Slovenia between 1992 and 1995. Strains were isolated in culture from skin biopsies of acrodermatitis chronica atrophicans lesions; in the case of one patient two separate acrodermatitis chronica atrophicans lesions were cultured. All 21 patients had clinically typical lesions with "classic" histopathology and high IgG antibody titers to B. burgdorferi sensu lato. Strains were characterized and typed by 16S ribosomal RNA-specific polymerase chain reaction and determination of their large restriction fragment patterns using pulsed-field gel electrophoresis of MluI-digested genomic DNA. Of the 22 isolates studied, 17 possessed the highly conserved MLa1 pattern characteristic of B. afzelii. The remaining five isolates possessed large restriction fragment patterns that were typical of B. garinii (MLg2, four isolates from three patients) and B. burgdorferi sensu stricto (MLb2, one isolate). The results of 16S ribosomal RNA-specific polymerase chain reaction were concordant with these species designations. These data show that B. afzelii is the predominant, but not the exclusive, etiologic agent of acrodermatitis chronica atrophicans.