To construct a DNA probe specific for protozoa that cause visceral leishmaniasis, we cloned Pst I fragments of Leishmania infantum genomic DNA into a Bluescript II SK® vector. A clone of 4.3 kb that contained a highly repetitive sequence was isolated and cut with three restriction enzymes: Hae III, Rsa I, and Sau 3A. After a new molecular cloning step, we isolated and sequenced a 140-basepair (bp) fragment. Two oligonucleotides were synthesized to be used as primers for a polymerase chain reaction. Using this probe, we detected an amount of DNA equivalent to one promastigote of L. infantum. This probe showed a high specificity; all protozoa tested that cause visceral leishmaniasis and L. major (one of the causative agents of Old World cutaneous leishmaniasis) showed a 100-bp amplified sequence, whereas other Leishmania strains showed a signal of a different size or else no signal. Moreover, no amplified sequence was obtained with other pathogenic parasites tested (Trypanosoma brucei, T. cruzi, Plasmodium falciparum, Pneumocystis carinii, and Toxoplasma gondii).