Molecular analysis of kinetoplast deoxyribonucleic acid (kDNA) minicircles has permitted the genotypic characterization of pathogenic isolates of Leishmania species. The apparent size in agarose gels of unit-length minicircles released by EcoRI digestion of kDNA networks is not conserved during speciation in this genus since the minicircles of strains and clones of L. major are smaller (710 base pairs, bp) than those found in certain strains of L. mexicana subspecies (820 bp), L. donovani (825, 865 bp) or L. tropica (900, 930 bp). EcoRI-cut minicircles within any one species of Leishmania are heterogeneous in mobility during electrophoresis in acrylamide gels. Schizodeme analysis of minicircles reveals a high degree of sequence divergence in kDNA with the degree of microheterogeneity varying between species. This sequence divergence allows the discrimination of closely related clones and strains within a given species. Southern blot hybridization reveals that overall minicircle sequence homology is conserved among clones and strains of one species (L. major or L. tropica) but not between different species. This property of minicircle DNA permits the use of kDNA probes as a species-specific diagnostic test for the identification of Leishmania isolates. The analysis of kDNA from two L. tropica strains isolated at 14 year intervals from a patient with leishmaniasis recidivans has shown that the two strains are closely related, suggesting that the individual suffered the cutaneous disease as a result of a resurgence of the same parasite which caused the initial infection. The differences in the properties of kDNA from the L. tropica and L. major strains studied support the taxonomic separation of L. tropica and L. major into distinct species.