We investigated the endemicity of Pseudomonas aeruginosa in intensive care units (ICUs) through analyses of surveillance cultures (from the rectum, stomach, oropharynx, and trachea; n = 1,089), and clinical cultures (n = 2,393) from 297 consecutive patients. Multiple isolates of P. aeruginosa (n = 353) were genotyped. Variables associated with acquisition of respiratory tract colonization (RTC) were tested in a risk factor analysis. The mean daily prevalence of colonization was 34%. On admission, 22 patients had intestinal colonization and 13 had RTC. Twenty patients acquired colonization in the intestinal and 24 in the respiratory tract. Forty-four different genotypes were found; 38 (86%) were isolated from individual patients only. In all, 37 patients had RTC with a total of 38 genotypes: 13 (34%) were colonized on admission, 9 (24%) acquired RTC with a novel genotype during a stay in the ICU, five (13%) acquired colonization from their intestinal tract and three (8%) were colonized via cross-acquisition. In eight patients (21%), no route could be demonstrated for colonization. Antibiotics providing P. aeruginosa with a selective growth advantage were associated with acquired RTC. Endemicity of colonization with P. aeruginosa is characterized by polyclonality, and seems to be maintained by continuous admittance of colonized patients and selection pressure from antibiotics rather than by cross-acquisition.