Seventy-six cases of visceral leishmaniasis (VL) were reported from the Hebron district of the West Bank, Palestine between 1993 and 2007. All cases were in children less than 9 years old (median age 2 years). The average number of cases was 5.06/year and the average annual incidence was 3.02/100000 children. Ribosomal internal transcribed spacer 1 (ITS1) PCR-RFLP was performed using DNA extracted from two cultures and 36 archived Giemsa-stained slides from VL patients. Leishmania infantum was revealed as the causative agent of VL in the focus. Isoenzyme analysis identified two isolates as zymodeme MON-1. A serological survey of 455 children screened for serum anti-Leishmania antibodies revealed 8.4% seropositivity. Seropositivity was highest for children in households of previous VL cases [odds ratio (OR) 7.5; 95% CI 3.17-17.61; P<0.001] and among people who had domestic dogs and/or other animals (OR 2.4; 95% CI 1.19-4.68; P=0.017). No difference was seen between males and females (P=0.073). A preliminary survey of sand fly distribution showed the abundance of two putative vector species: Phlebotomus syriacus (45%) and Ph. tobbi (10%). The focus of VL in Hebron district was shown to follow the epidemiological pattern of paediatric disease characteristic of the Mediterranean region.