The spectrum of organisms responsible for lower respiratory tract infection in chronically ventilated neonates is poorly defined. During an 18-month period 63 infants with a respiratory deterioration defined as an increase in fractional inspired O2 concentration > or = 20% and/or mean airway pressure > or = 3 cm H2O were evaluated for pulmonary infection. These infants were compared with 58 stable control ventilated infants. Tracheal aspirates for culture and Gram stain were taken from both groups and were cultured for bacteria, viruses, Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis. In addition each infant had complete blood counts with differential and chest roentgenograms evaluated. Positive tracheal aspirates defined as a heavy growth of a single or two bacterial organisms, and/or any growth of virus, Chlamydia and U. urealyticum were found in 23 of 63 study patients and 20 of 58 controls (P > 0.05). The most frequent isolate in both groups was U. urealyticum. Chest radiographs were positive (new changes, particularly atelectasis and infiltrates) more frequently in the study group than in controls, but complete blood count and tracheal aspirate Gram-stained smears were not helpful in discerning colonization from infection. We conclude that positive tracheal aspirates occur with equal frequency among infants with a clinical suspicion of lower respiratory tract infection and in "well" controls. Chest roentgenogram may be a useful adjunctive test to discriminate between colonization and lower respiratory tract infection.