This study assessed the recovery rates of Gram-negative bacilli from stored endotracheal aspirates frozen with and without glycerol. Samples frozen with glycerol showed a significant difference in isolate recovery, 89.7% versus 69.2% (P = 0.02). This study demonstrates that it is possible to achieve high recovery rates of potentially pathogenic organisms from endotracheal aspirates when stored with glycerol, thus broadening the scope of active surveillance cultures for both clinical and research purposes.