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Xenodiagnosis of Leishmania infection in hares (Lepus granatensis) from a focus of human leishmaniasis in Fuenlabrada at southwestern Madrid region (Spain) proved that they are infective to Phlebotomus perniciosus. Molecular characterization of isolates obtained from sand flies infected after xenodiagnosis demonstrates that hares were infected by Leishmania infantum. This is the first evidence of the transmission of L. infantum from hares to sand flies. Moreover the results confirm the role that these animals can play as wild reservoirs of leishmaniasis for the recent outbreak of visceral leishmaniasis in Madrid.