An endemic focus of American visceral leishmaniasis (AVL) in eastern Venezuela has been evaluated in terms of patients (n = 48), immunologic reactivity to Leishmania in household contacts (n = 187) and neighborhood controls (n = 170), detection of Leishmania (L. donovani complex) in dogs and wild animals by the polymerase chain reaction (PCR) and characteristics of the sandfly population. The male:female ratio of patients was 1.18:1; 89.6% were < or =12 years old. Serologic reactivity was significantly higher in household contacts than in controls (P = 0.0008), as was the size of leishmanin reactions in contacts < or =10 years of age (P = 0.0141). Leishmania donovani complex-specific PCRs were positive in dogs, an opossum (Didelphis marsupialis), and a black rat (Rattus rattus). Lutzomyia longipalpis and Lu. evansi, both implicated in the transmission of AVL, were identified among the 386 sand flies examined. These observations provide the bases for an active control program as well as further studies of reservoirs and vector-host relationships in this area.