In this study we developed and tested an adenosine triphosphate (ATP) bioluminescence-based protocol for rapid characterization of bioaerosol sampling devices when collecting bacterial aerosols. Initial calibration of the method was performed using commercially available ATP assays to quantify known bacterial biomass in liquid samples. Here, the relative luminescence units (RLUs) indicative of total ATP content was plotted against total bacterial counts obtained using acridine orange direct count epifluorescence microscopy. The developed calibration curves were used to evaluate the collection performance of a BioSampler (SKC Inc., Eighty Four, PA) when sampling Pseudomonas fluorescens and Bacillus subtilis bacteria at three different sampling flow rates. The average collection efficiency for P. fluorescens and B. subtilis over all flow rates was 69% and 54%, respectively. Collection efficiencies determined by the ATP method for both bacteria at all flow rates agreed well (within 5%) with the acridine orange direct counts. Further, the ATP-based method allowed insights into the losses and fate of particles inside the sampling device. It was determined that a majority of losses occur in sampler's inlet. Such evaluation of losses is very difficult to perform using microscopy or culture-based techniques. Thus, the ATP-based technique could be reliably used as a tool for rapid and detailed evaluation of bioaerosol sampling devices. © 2008 Elsevier Ltd. All rights reserved.