A restriction fragment from a cloned kinetoplast minicircle DNA has been shown to be diagnostic for Leishmania major. This 402-bp TaqI fragment has been used routinely (as a radiolabelled probe) to detect 10(4) parasites in simple dot blots, both experimentally and in epidemiological surveys. It positively identified all stocks of L. major tested (including all six known zymodemes) and showed very low homology to kinetoplast DNA (kDNA) and chromosomal DNA of Leishmania infantum and Leishmania tropica, two species commonly isolated from patients and wild hosts within foci of L. major in the Old World. DNA sequence analysis of a minicircle of L. major is reported for the first time, and it is demonstrated that this species shares with Leishmania aethiopica, Sauroleishmania tarentolae and several species of Trypanosoma a region of conserved sequence that is involved in DNA replication, a process that could present targets for selective chemotherapeutic attack. Sequence and restriction fragment analyses have indicated the difficulties of selecting species-specific sequences from kDNA which, even in the same parasite clone, contains several predominant minicircle classes, not all of which contain diagnostic sequences.