An epidemiological investigation was performed to evaluate the prevalence of anti-leptospiral agglutinins within a selected population: the railway workers of the Italian Railway Compartments of Bologna and Venice. Leptospira infection prevalence was found to be 10.38%, which represents the highest frequency ever reported in Italy. There was a significantly higher prevalence of agglutinins among the staff of the Railway Compartment of Venice. A clear prevalence of the bratislava serovar was registered, followed by the australis serovar and the icterohaemorrhagiae serovar. The influence of different kinds of railway work and that of supposed risk factors, such as hobbies and housing conditions, together with the influence of the ABO blood groups on the distribution of Leptospira agglutinins, are discussed.