A prospective study was initiated in an intensive care unit (ICU) where extended-spectrum beta-lactamase-producing enterobacteriaceae (ESBLPE) were endemic. From July 1990 to July 1991, patients hospitalized for > or = 5 days were screened for ESBLPE acquisition by means of weekly rectal sampling and clinical cultures. Baseline characteristics and various ICU procedures in 62 cases of ESBLPE were compared with those for 205 patients without ESBLPE, with use of Cox's model. Risk for acquiring ESBLPE (Klebsiella pneumoniae in most cases) increased during the ICU stay, from 4.2% in the first week to 24% in the fourth week. Baseline characteristics were not different between the two groups. Urinary catheterization (P = .04) and arterial catheterization (P = .03) were independent risk factors for acquiring ESBLPE and probably reflected frequency of health care manipulations. The first site of ESBLPE acquisition was the digestive tract in 58 of the 62 patients; 28 infections developed in 22 patients, and these followed or occurred simultaneously with rectal colonization in 18 of those 22. DNA macrorestriction analysis suggested that the same strain was responsible for most cases. In conclusion, ESBLPE acquisition depends on length of stay in the ICU and the use of invasive procedures. Colonization is a prerequisite for infection.