The aim of this study was to investigate the patterns and dynamics of the microbiota in the airways of ventilated patients. Seventy-four mechanically-ventilated patients were recruited consecutively, and oropharyngeal, tracheal and bronchoalveolar (BAL) fluid specimens were collected 48 h after intubation, and every 72 h thereafter until the patient was extubated or a total of five sample sets had been collected. Ventilator-associated pneumonia (VAP) pathogens were identified, quantified and genotyped. Microbial findings were highly correlated both between airway locations and over time when samples were taken no more than 72 h apart. If no VAP pathogen was present in the oral flora, it was unlikely to be found in a lower airway sample; i.e., the positive predictive value of the oropharyngeal sample was 0.73 (95% CI 0.67-0.80), and the negative predictive value was 0.95 (95% CI 0.92-0.99). Colonisation with Enterobacteriacae, non-fermentative bacteria and Staphylococcus aureus was monoclonal in the airways and over time, whereas colonisation with microbes normally found in the oropharynx, i.e., Haemophilus influenzae, Haemophilus parainfluenzae and Streptococcus pneumoniae, was polyclonal. When antibiotics were used, the chance of recovering VAP pathogens from all sampling sites was reduced three-fold. No correlation was observed between a bacterial count of > or =10(4) CFU/mL in BAL fluid and chest X-rays compatible with VAP.