Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca²⁺ signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at 60 mV, ACh (0.1-10 µM) induced a concentration-dependent inward current, which, in ~50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 µM). Exposure to acrolein (0.2 µM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 µM) concentration-induced initial peak of current (318.8 ± 28.3 vs. 251.2 ± 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 µM), the frequency at which subsequent peaks occurred was significantly increased (13.2 ± 1.1 vs. 8.7 ± 2 min¹; n = 20, P < 0.05). ACh-induced current was identified as a Ca²⁺-activated Cl current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca²⁺ release, did not alter caffeine-induced transient membrane currents (595 ± 45 and 640 ± 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca²⁺ concentration response and that the protective role of inhibitors of Cl channels in air pollutant-induced airway hyperresponsiveness should be examined.