A system for the detection of a Borrelia burgdorferi (Bb)-specific gene segment in formalin-fixed, paraffin-embedded skin lesions is described. A nested polymerase chain reaction technique is used to selectively amplify in vitro a short segment of a Bb-specific gene recently described by Rosa et al. (J Infect Dis 1989: 160: 1018). The design of oligonucleotide primers for the amplification of a relatively short gene segment allows the successful analysis of DNA which has been altered by fixation in formalin. Using this technique, Bb-specific DNA was clearly identified in 8 of 12 specimens of erythema chronicum migrans and in 1 case of lymphadenosis benigna cutis. These skin lesions are known to represent cutaneous manifestations of Lyme disease. Negative control reactions, using DNA from borrelial strains not related to Lyme disease, were negative. The system enables the dermatopathologist to identify Bb in routinely fixed clinical specimens and allows the rapid analysis of various skin diseases for which an association with Bb so far has only been hypothesized.