Background: Clinical acumen alone is unreliable in establishing a diagnosis of ventilator-associated pneumonia (VAP) and controversy exists over which diagnostic tools should be utilized to confirm a clinical suspicion of VAP. The purpose of this study was to determine the reliability of blind protected specimen brush (PSB) sampling in the diagnosis of VAP and if bilateral PSB sampling is necessary. Methods: Prospective study comparing blind PSB sampling with bronchoscopic directed PSB sampling in thirty-four consecutive SICU patients with a clinical suspicion of VAP. All patients underwent blind PSB sampling followed by bronchoscopic directed contralateral PSB sampling. Results: Twenty-four of 34 patients (71%) were diagnosed to have VAP. The concordance rate between blind and directed PSB samples was 53% (18/34). When blind PSB was positive (15/34), the contralateral sample yielded a different microorganism in three patients (9%). When blind PSB was negative (19/34), infection was present in the contralateral lung in nine patients (26%). Blind PSB sampling alone was inaccurate in 35% of patients. Conclusions: The low concordance between blind and directed PSB suggests the need to sample both lung fields. Bilateral PSB sampling can identify unsuspected pathogenic microorganisms in the contralateral lung.