Between 1988 and 1998, 258 Leishmania strains from patients infected with HIV were characterized by iso-enzyme electrophoresis at the Istituto Superiore di Sanità (ISS) in Rome. Most (227) of the isolates came from 80 Italian patients with visceral leishmaniasis (VL), the rest from cases of Leishmania/HIV co-infection in other Mediterranean countries. Every strain was found to be Leishmania infantum. In Italy, 19 zymodemes of L. infantum were identified, broadly divided into three groups. Over 50% of the Italian patients were infected by the commonest agent of Mediterranean VL in HIV-negative individuals (zymodeme MON-1) whereas the remaining patients were infected by two distinct groups of zymodemes: one usually causing simple, self-resolving cutaneous leishmaniasis in HIV-negative patients, the other consisting of agents detected, so far, only in HIV-positive subjects. This last group, consisting of seven zymodemes, showed re-assortment patterns within electromorphs frequently observed in dermotropic L. infantum zymodemes. A similar grouping was also observed in the isolates from other Mediterranean countries. Basing on general data recorded at the ISS over the last 20 years, accurate identification of the geographical origin of the zymodemes was attempted by careful analysis of the patients' histories, using the iso-enzyme electromorphs as geographical markers. Furthermore, a polymorphism index (no. of zymodemes/no. of patients) was defined for each Leishmania species and geographical region, and used to assess the level of L. infantum zymodeme heterogeneity in Italy, before and after stratification by HIV status. The greatest zymodeme heterogeneity was found in Sicily (southern Italy), with nine zymodemes identified among only 30 HIV-positive patients.