Pseudomonas aeruginosa is one of the leading nosocomial pathogens. In hospitals, organisms are commonly recovered from moist environments. To determine the reservoir and population dynamics of particular strains, highly discriminative typing methods are required. Hybridization of enzymatically restricted P. aeruginosa DNA with two gene probes led to the identification of infecting and colonizing strains prevalent over a 5-month period in a neonatal intensive care unit. Four genotypically distinct strains were repetitively isolated from tap water from several faucets on the ward. P. aeruginosa isolates recovered from tap water on adjacent wards supplied by the same water system had different genotypes, while samples taken from the mains were negative for the organism. Serotyping of O antigens showed variable reproducibility and could not elucidate the strain-specific reservoirs. It is concluded that organisms are transmitted horizontally between faucets and prevail in reservoirs for prolonged periods.