Leishmania parasites from animals, man or insect vectors were characterized by the gel electrophoresis of restriction endonuclease enzyme-produced mitochondrial (kinetoplast) DNA (kDNA) fragments and/or by DNA-DNA hybridization with 32P-labelled cloned, or uncloned, kDNA fragment probes from type isolates. The electrophoretic separation of kDNA fragments is a sensitive method for detecting genetic similarities and differences among Leishmania. Parasites with similar kDNA restriction fragment patterns belong to the same schizodeme and schizodeme analysis is useful for studying Leishmania populations. Cloned, species-specific kDNA probes detected Leishmania in sandflies and in liver, spleen or blood preparations from infected animals. Cloned DNA probes also hybridized to immobilized kDNA from in vitro cultivated promastigotes and detected as few as 100 parasites in a species-specific manner. Sensitive DNA hybridization probes should be useful in research on the immunology, chemotherapy or epidemiology of animal and human leishmaniasis.