In order to investigate the possible role of dog fleas in the transmission of trypanosomatids, ectoparasites were removed from 59 dogs testing positive for canine zoonotic visceral leishmaniasis according to the indirect fluorescent antibody test (IFAT). Of the fleas collected, 4/207 (1.9%) showed the presence of promastigotes in smears stained by Giemsa, whilst 43/144 (29.9%) exhibited positive polymerase chain reaction (PCR) amplification assays for Leishmania DNA. Fleas (409) from 9 Leishmania chagasi-infected dogs, each hosting more than 20 fleas per animal, were macerated and administered by peritoneal injection or orally to 36 hamsters. After 6 months, the 30 surviving hamsters were sacrificed and liver and spleen fragments were removed for PCA assay and to produce imprint smears, whilst blood samples were subjected to IFAT assay. Sixteen hamsters tested positive for Leishmania infection, 14 on the basis of PCR amplification and four by IFAT assay (two animals testing positive in both assays). Of the infected hamsters, 11/16 (68.7%) had been infected peritoneally and 5/16 (31.2%) orally. The imprint smears for all animals were, however, negative. Since both PCR and IFAT could present cross-reactivity for Leishmania and Leptomonas, the possibility of oral transmission of L. chagasi by fleas cannot be proven unambiguously even though the hamsters developed infection.