In hospital and other health care facilities, contamination of water systems by potentially infectious microorganisms, such as bacteria, viruses and protozoa, is a source of nosocomial infections, which may originate fromcolonization of water pipes, cooling towers, spa pools, taps, showers and water supplies. Objective. The study focuses on the occurrence of Legionella spp., free-living amoebae and non-fermenting Gram-negative microorganisms in a University hospital water system located in the town of Messina (Sicily, Italy), which had never been examined previously. From January 2008 - March 2009, hot tap water samples were collected from 10 wards. Legionella spp. recovered on selective culture medium were identified by microagglutination latex test; free-living amoebae were cultured using Escherichia coli as a food source. Non-fermenting Gram negative microorganisms were identified by API 20 NE strips. Legionella spp. were found in 33.33% of the samples. L. pneumophila serogroup 1 was recovered from the Laboratory Diagnostic and Anaesthesia-Neurology Wards, with a peak of 3.5 × 10(4) cfu/L in May 2008. L. pneumophila serogroups 2-14 were found in the Othorhinolaryngology, Pathologic Anatomy, Paediatrics and Surgery Wards, and peaked (4 × 10(4) cfu/L) in April 2008. Pseudomonadaceae and Hyphomycetes were also detected. Legionella spp. were recovered from samples positive for non-pathogenic amoebae Hartmannella spp. This first study of a Messina hospital water system suggested potential health risks related to the detection of Hartmannella spp., as reservoirs for Legionella spp., and Pseudomonas aeruginosa, a Gram negative non-fermenting bacterium frequently causing nosocomial pneumonia. The urgent need for monitoring programmes and prevention measures to ensure hospital water safety is stressed.