In Triton-skinned phasic ileal smooth muscle, constitutively active recombinant p21-activated kinase (PAK3) has been shown to induce Ca²⁺-independent contraction, which is accompanied by phosphorylation of caldesmon and desmin (Van Eyk JE, Arrell DK, Foster DB, Strauss JD, Heinonen TY, Furmaniak-Kazmierczak E, Cote GP, and Mak AS. J Biol Chem 273: 23433-23439, 1998). In the present study, we investigated whether PAK has a broad impact on smooth muscle in general by testing the hypothesis that PAK induces Ca²⁺-independent contractions and/or Ca²⁺ sensitization in tonic airway smooth muscle and that the process is mediated via phosphorylation of caldesmon. In the absence of Ca²⁺ (pCa > 9), constitutively active glutathione-S-transferase-murine PAK3 (GST-mPAK3) caused force generation of Triton-skinned canine tracheal smooth muscle (TSM) fibers to ~40% of the maximal force generated by Ca²⁺ at pCa 4.4. In addition, GST-mPAK3 enhanced Ca²⁺ sensitivity of contraction by increasing force generation by 80% at intermediate Ca²⁺ concentrations (pCa 6.2), whereas it had no effect at pCa 4.4. Catalytically inactive GST-mPAK3^K297R had no effect on force production. Using antibody against one of the PAK-phosphorylated sites (Ser⁶⁵⁷) on caldesmon, we showed that a basal level of phosphorylation of caldesmon occurs at this site in skinned TSM and that PAK-induced contraction was accompanied by a significant increase in the level of phosphorylation. Western blot analyses show that PAK1 is the predominant PAK isoform expressed in murine, rat, canine, and porcine TSM. We conclude that PAK causes Ca²⁺-independent contractions and produces Ca²⁺ sensitization of skinned phasic and tonic smooth muscle, which involves an incremental increase in caldesmon phosphorylation.