On the basis of partial amplification of a cloned fragment of kDNA of Leishmania infantum which is specific for this species, we developed a PCR-ELISA technique which avoids the problems associated with classical diagnostic techniques. This technique was tested on 33 L. infantum strains from 19 different zymodemes, which were recognized equally. It was also used on human and canine clinical samples. PCR-ELISA has a higher sensitivity than the other techniques used (IFAT, parasite cultures, optical microscopy of stained samples) and permits detection of a minimum of 0.1 promastigotes or 1 fg of genomic DNA. PCR-ELISA can be used to diagnose human cutaneous leishmaniasis using material obtained by scraping the lesion margin, and human visceral leishmaniasis in HIV(+) individuals and canine leishmaniasis with peripheral blood samples. The presence of L. infantum in dogs with low antibody titres with IFAT technique (20 and 40) was demonstrated indicating that seroprevalence data from epidemiological studies underestimate the true rates of infection.