OBJECTIVE: To determine the potential of laboratory services in identifying cross-transmission of multiresistant Gram-negative bacilli (MR GNB) and Staphylococcus aureus in adult intensive care units by routine typing of clinical isolates. METHODS: Over a 12-month period, isolates with indistinguishable PCR fingerprints were traced back to the source patients and their epidemiologic relationships were investigated. Possible episodes of cross-transmission were ascertained, and the validity of antibiograms in identifying the same cluster assessed. RESULTS: Of 3503 specimens received by the microbiological laboratory during 5372 patient days, 1295 cultures showed bacterial growth. Of these, 132 were primary isolates of MR GNB and 92 were primary isolates of S. aureus. Thirty-two MR GNB isolates (24%) shared fingerprints with one or more other isolates. Indistinguishable isolates from epidemiologically related patients suggested 17 episodes of cross-transmission. The positive and negative predictive values of antibiogram-based identification of these episodes were 19% and 72% respectively. S aureus displayed limited genetic diversity. The two most frequent genotypes contained 19 and 16 isolates, of which the majority appeared to be epidemiologically unrelated. CONCLUSIONS: Endemic transmission of MR GNB occurs mainly between two patients and remains unrecognized by conventional laboratory investigation. Rapid genetic typing methods identify patients involved in cross-transmission and give an insight into the population dynamics of MR GNB on adult intensive care units.