Leishmania infantum, the causative agent of visceral leishmaniasis (VL) in Brazil, is spread mostly by the bite of the sand fly Lutzomyia longipalpis (Lutz & Neiva). We trapped sand flies in endemic neighborhoods near Natal, Brazil, where cases of human and dog VL were documented. Amplification of species-specific cytochrome b (Cyt b) genes by polymerase chain reaction revealed that sand flies from rural and periurban areas harbored blood from different sources. The most common source of bloodmeal was human, but blood from dog, chicken, and armadillo was also present. We tested the preference for a source of bloodmeal experimentally by feeding L. longipalpis F₁ with blood from different animals. There were significant differences between the proportion of flies engorged and number of eggs laid among flies fed on different sources, varying from 8.4 to 19 (P < 0.0001). Blood from guinea pig or horse was best to support sand fly oviposition, but human blood also supported sand fly oviposition well. No sand flies fed on cats, and sand flies feeding on the opossum Monodelphis domestica Wagner produced no eggs. These data support the hypothesis that L. longipalpis is an eclectic feeder, and humans are an important source of blood for this sand fly species in periurban areas of Brazil.