Twenty-two black rats (Rattus rattus) were captured in houses where parasitologically confirmed cases of human visceral leishmaniasis had been recorded in Al-Arda Emara, Gizan province, south-west Saudi Arabia. Four of the rats were found to be infected with Leishmania; isoenzyme characterization showed that 3 were infected with L. donovani sensu lato zymodeme LON42 and the fourth with L. infantum zymodeme LON49. L. donovani s.l. LON42 has also been isolated from human visceral leishmaniasis patients living in this area, while dogs, but not humans, have been found to be infected with L. infantum LON49 in this part of Saudi Arabia.