A 196 bp fragment of cloned kinetoplast (k) DNA of Leishmania infantum has been sequenced and shown to be diagnostic for this species. The DNA from 104–105 promastigotes was routinely and specifically detected in crude dot blots of whole cells lysed in situ, and there was no cross-hybridization with 106 cells of L. major, L. tropica, L. killicki, L. aethiopica, L. braziliensis and L. mexicana. A low degree of hybridization was found with L. donovani and L. chagasi. This probe will be of particular use in Mediterranean countries, such as Italy, in which dermotropic zymodemes of L. infantum can be difficult to isolate and grow in numbers sufficient for isoenzyme typing.