To elucidate the sources of Pseudomonas aeruginosa on a surgical intensive care unit, rDNA restriction fragment length polymorphism analysis (ribotyping) was applied to analyze strains isolated during a 4-month prospective study. Samples included 1635 from 153 patients, 2463 from 97 staff members, and 581 from the environment. Only 18 patients were colonized. Isolation from their animate and inanimate environment was very low, with 3 and 2 samples, respectively, being positive. Samples from tap water were negative. Ribotyping could easily distinguish 16 different digest patterns with identical follow-up isolates of the same patient. Horizontal transmission occurred only twice. The discriminatory power of ribosomal DNA in differentiating strains was dependent on the restriction enzymes used; among eight different enzymes, PvuII was the most sensitive, producing 15 different patterns. Ribotyping showed high sensitivity in typing P. aeruginosa isolates and confirmed that colonization occurs from endogenous rather than from exogenous sources.