Inhaled aerosols have wide-ranging applications in the treatment and diagnosis of respiratory diseases. Methacholine challenge testing (MCT) is a diagnostic test frequently used to evaluate airway hyper-reactivity. We hypothesize that a significant fraction of the inhaled dose of methacholine is exhaled during treatment, given the small droplet sizes produced by nebulizers traditionally used for MCT. Here, an in vitro – in silico approach was developed to predict respiratory tract deposition achieved with various nebulizers proposed for use with MCT.Emitted doses, particle sizes, and the temperature and humidity of the nebulized airstream were experimentally measured for three nebulizers (RX160, Roxon Meditech; Hudson RCI Micro Mist [HRCI], Teleflex; AirLife Misty Max 10 [MM10], CareFusion) at methacholine chloride concentrations of 0.0625, 1, and 16 mg/mL in 0.9% saline solutions. Emitted doses at a concentration of 1 mg/mL were measured to be 42.0 (SD 5.1) μg from the RX160 over 120 s, 96.3 (SD 33.7) μg from the HRCI over 60 s, and 162.3 (SD 38.4) μg from the MM10 over 60 s. For a typical adult tidal inhalation, the inhaled dose was found to be half of the emitted dose. Our hygroscopic lung deposition model predicted considerable condensational growth within the respiratory tract for aerosols used with MCT. For the 1 mg/mL methacholine chloride solution, the fraction of the inhaled dose predicted to deposit in the lungs was 0.40 (8.3 μg) for the RX160, 0.62 (29.6 μg) for the HRCI, and 0.60 (48.7 μg) for the MM10. Predicted exhaled dose fractions were greatest with the RX160 (0.60) and similar for the HCRI (0.34) and MM10 (0.36). Hygroscopic modeling thus suggests that the exhaled dose obtained during methacholine challenge testing is considerable, with the characteristics of the nebulizer influencing the relative proportion of the dose that is exhaled.