Aims: This study was designed to define the extent of water contamination by Legionella pneumophila of certain Italian hotels and to compare quantitative real-time PCR with the conventional culture method. Methods and Results: Nineteen Italian hotels of different sizes were investigated. In each hotel three hot water samples (boiler, room showers, recycling) and one cold water sample (inlet) were collected. Physico-chemical parameters were also analysed. Legionella pneumophila was detected in 42% and 74% of the hotels investigated by the culture method and by real-time PCR, respectively. In 21% of samples analysed by the culture method, a concentration of > 104 CFU l-1 was found, and Leg. pneumophila serogroup 1 was isolated from 10 center dot 5% of the hotels. The presence of Leg. pneumophila was significantly influenced by water sample temperature, while no association with water hardness or residual-free chlorine was found. Conclusions: This study showed a high percentage of buildings colonized by Leg. pneumophila. Moreover, real-time PCR proved to be sensitive enough to detect lower levels of contamination than the culture method. Significance and Impact of the Study: This study indicates that the Italian hotels represent a possible source of risk for Legionnaires' disease and confirms the sensitivity of the molecular method. To our knowledge, this is the first report to demonstrate Legionella contamination in Italian hotels using real-time PCR and culture methods.