Borrelia burgdorferi sensu lato, the aetiological agent of Lyme disease, has been subdivided into three species: B. burgdorferi sensu stricto, B. garinii and B. afzelii. We and other authors have hypothesized an association between the three species of B. burgdorferi sensu lato and some of the different clinical manifestations of Lyme disease. In order to demonstrate this hypothesis, we analysed twenty-nine isolates cultured from patients with different symptoms. The method used was multilocus enzyme electrophoresis: twelve genetic loci were characterized on the basis of the electrophoretic mobility of their products, and twenty-eight distinctive allele profiles (electrophoretic types) were distinguished, among which mean genetic diversity per locus was 0.649. Cluster analysis of a matrix of genetic distances between paired electrophoretic types revealed three primary divisions separated at genetic distances greater than 0.7 and corresponding to the three species of B. burgdorferi sensu lato. Ten strains obtained from skin of patients with erythema chronicum migrans (the primary stage of the disease) were assigned to the three different species. All the six strains isolated from patients with acrodermatitis chronica atrophicans were of the species B. afzelii, which was not found to be associated with another chronic manifestation of Lyme disease. Arthritis was caused prevalently by B. burgdorferi sensu stricto, and neuroborreliosis by B. burgdorferi sensu stricto and B. garinii. In conclusion, our results confirm the association between some of the different chronic manifestations of the disease and the species of B. burgdorferi sensu lato.