Accessory devices reduce common problems with metered-dose inhalers (MDIs), namely high oropharyngeal deposition of aerosol and incoordination between actuation and inhalation by the patient. The objective of this study was to systematically compare the performance of various accessory devices in vitro. MDIs were tested alone or in combination with four spacers (Toilet paper roll, Ellipse, Optihaler, Myst Assist) and five holding chambers (Aerochamber, Optichamber, Aerosol Cloud Enhancer, Medispacer, and Inspirease). An Anderson cascade impactor was used to measure aerosol mass median aerodynamic diameter (MMAD) and fine particle dose (MMAD < 4.7 microm). In separate experiments, the influence of asynchronous MDI actuation on drug delivery was determined with a simulated spontaneous breathing model. Compared with the MDI alone, all of the accessory devices reduced aerosol MMAD and increased lung-throat ratio (fine particle dose/throat impaction; p < 0.05 for both parameters). The fine particle dose of albuterol was 40% higher with the Ellipse (p < 0.01), was equivalent with the Toilet Paper Roll, Aerochamber, Optichamber, and Medispacer, and was 33-56% lower with the Optihaler, Myst Assist, Aerosol Cloud Enhancer, and Inspirease (p < 0.03). MDI actuation in synchrony with inspiration produced highest drug delivery; when MDI actuation occurred 1-sec before inspiration or during exhalation, decrease in drug delivery with holding chambers (10-40% reduction) was less than that with spacers (40-90% reduction). Accessory device selection is complicated by variability in performance between devices, and in the performance of each device in different clinical settings. In vitro characterization of a MDI and accessory device could guide appropriate device selection in various clinical settings.