The adapter protein paxillin has been implicated in the regulation of cytoskeletal organization and cell motility. Paxillin undergoes tyrosine phosphorylation in response to the contractile stimulation of smooth muscle, and the depletion of paxillin by antisense inhibits smooth muscle contraction. In the present study, acetylcholine (ACh)-stimulation of tracheal smooth muscle tissues increased paxillin phosphorylation at tyr-31 and tyr-118 by three- to fourfold. The role of tyr-31 and tyr-118 phosphorylation of paxillin in smooth muscle was evaluated by introducing plasmids encoding wild type paxillin or paxillin mutants F31, F118 or F31/118 (phenylalanine substitution at tyrosine sites 31, 118) into tracheal smooth muscle strips by reversible permeabilization, and incubating the tissues for 2 days. The expression of recombinant proteins was confirmed by immunoblot and immunofluorescence analysis. Expression of the paxillin mutants F31, F118 or F31/118 inhibited the contractile response to ACh stimulation but did not inhibit the increase in myosin light chain phosphorylation. The expression of wild type paxillin had no significant affect on force or myosin light chain phosphorylation. ACh stimulation reduced G-actin/F-actin ratio in tissues expressing wild type paxillin; whereas the agonist-induced decrease in G-actin/F-actin was inhibited in strips expressing paxillin mutant F31/118. The paxillin mutant F31/118 showed a marked decrease in their interaction with the SH2/SH3 adaptor protein CrkII but not with vinculin or focal adhesion kinase. We conclude that paxillin phosphorylation at tyr-31 and tyr-118 regulates active tension development during contractile stimulation. Paxillin phosphorylation at these two sites may be important in regulating actin filament dynamics and organization during smooth muscle contraction.