Findings from a seroepidemiological survey of Leishmania infantum (LI) infection are presented. Among residents in the province of Rome, 2.7% (4.4% in the city of Rome and 1% in the suburbs) of 374 adults were positive for specific LI antibodies in indirect haemoagglutination studies. Among 217 adult residents of Caltanissetta province (Sicily), 4.1% were positive for LI antibodies (4.2% in Caltanissetta city and 4% in the suburbs). Serological tests for LI antibodies were also carried out on stray dogs captured in Rome. 5% of them resulted positive. Our findings show that LI infection is moderately present in both Rome and Caltanissetta, the latter being an area of known endemicity. According to our data, LI infections are usually asymptomatic: none of the positive subjects had a history of visceral leishmaniasis, and none of the seropositive dogs showed any sign or symptom correlated to leishmaniasis.