We investigated the mechanism of mucosal modulation of airway smooth muscle contraction by measuring concentration- and length dependencies of carbachol-induced active stress and myosin phosphorylation in mucosa-intact and mucosa-free bovine tracheal smooth muscle. The concentration dependencies of carbachol-induced active stress in mucosa-intact and mucosa-free smooth muscles were significantly different in maximum but not in half-maximal concentration (EC50). Similar mucosa-dependent difference in maximum was also observed in the concentration dependence of carbachol-induced myosin phosphorylation. As a result, the myosin phosphorylation-active stress relations in mucosa-intact and mucosa-free smooth muscles were not significantly different. Length dependence of carbachol-induced active stress was significant in mucosa-intact smooth muscle, and accompanied by significant length dependence of myosin phosphorylation. These results suggest that the primary effect of mucosal modulation is inhibition of myosin light chain phosphorylation without uncoupling of active stress from myosin phosphorylation in airway smooth muscle.