Colonization of the lower respiratory tract by enteric Gram-negative bacilli (EGNB) has been a frequent finding in patients with long-term tracheostomies; however, the association of hospitalization and certain features of serious illness with this phenomenon has not been clearly established. Because such factors can render the oropharynx more susceptible to EGNB colonization, we sought to discover whether they can also have this effect on the tracheobronchial tree and its microflora. Thus, we collected serial paired culture samples from these two mucosal sites in 15 subjects with long-term tracheostomies and examined patterns and rates of colonization and related these findings to clinical parameters. In 49 sets of cultures, we found that EGNB (especially Pseudomonas species) were present in significantly fewer upper-airway cultures (36.7 percent) than lower-airway cultures (75.5 percent) (p = 0.009). At the tracheobronchial site, seven subjects had persistent EGNB colonization, all with Pseudomonas species, while only one subject had this finding at the oropharyngeal site (p = 0.015). Patients with persistent tracheobronchial colonization were more ill than those without this finding. They were treated with higher doses of prednisone (p = 0.06), received antibiotics more often, and developed purulent tracheobronchitis more often (100 percent vs 25 percent) than patients without persistent colonization. In addition, in the month following the culture survey, four subjects developed pneumonia, and three of these had previous persistent tracheobronchial colonization.(ABSTRACT TRUNCATED AT 250 WORDS)