Rationale: The ability to determine the respiratory microbiome from bronchoalveolar lavage (BAL) is controversial due to oral contamination during bronchoscopy. To determine if processing BAL improved the ability to distinguish lung from oral wash microbiomes, we compared the oral and lung microbiome from whole and acellular BAL in 34 subjects. Methods: Oral wash samples and bronchoscopy with BAL were performed. After removing an aliquot of whole unprocessed BAL, the remaining was centrifuged at 1,500 rpm for 10 minutes and the supernatant harvested as acellular BAL. DNA was isolated from 2 ml of oral wash and 5 ml of acellular and whole BAL using the Qiagen DNeasy kit (Qiagen, Valencia, CA). DNA encoding 16s ribosomal RNA was amplified using NEB Phusion enzyme (NEB, Ipswich, MA) and degenerate of eubacterial primers that amplify the variable regions 1 through 3. DNA sequencing was performed using 454 sequencing. Results: The amount of genomic DNA isolated from whole BAL was 100-fold higher compared with acellular BAL. Despite this, the number of high-quality genera bacterial reads (RDP classifier > 90%) was identical between the two groups (whole BAL, 4,903 ± 3,209; acellular BAL, 4,245 ± 2,632). Bray-Curtis distances between oral wash and acellular BAL were significantly higher than the distance between whole BAL and oral wash (P = 0.000035), indicating acellular BAL was significantly more different from oral wash compared with whole BAL. Visually, the overlap between oral wash and whole BAL can be seen on the dendogram in Figure 1 (BA = acellular BAL; BW = whole BAL; OR = oral wash). [Figure: see text] Conclusions: The difference between acellular BAL and oral wash is significantly greater than the difference between whole BAL and oral wash. We suggest this represents greater contamination of whole BAL by upper airway organisms. We speculate acellular BAL provides a better representation of the true lower respiratory tract microbiome.