Characterization of pulmonary function parameters in mice will facilitate the dissection of genetic mechanisms underlying airway hyperresponsiveness. We evaluated acetylcholine (ACh)-induced respiratory system resistance (Rrs) and elastance (Ers) in A/J and C3H/HeJ mice and compared these results with the previously used airway pressure-time index (APTI). A low-dead-space ventilatory system was designed to ventilate anesthetized mice with constant inspiratory flow. The end-inflation occlusion method was used to measure Rrs and Ers at baseline and after intravenous ACh (12.5–75.0 micrograms/kg) challenge. ACh induced a dose-dependent rise in Rrs and Ers in A/J mice, whereas minimal changes were observed in C3H/HeJ mice. A/J mice had a higher baseline Rrs, yet the response to ACh was independent of baseline Rrs. Additionally, sequential ACh challenges led to augmented responses. Rrs, Ers, and APTI were strongly correlated, and each was useful to detect differences in interstrain cholinergic-induced airway responsiveness. The Rrs detected the smallest differences between the strains of mice studied.