The intermediate filament protein vimentin has been shown to be required for smooth muscle contraction. The adapter protein p130 Crk-associated substrate (CAS) participates in the signaling processes that regulate force development in smooth muscle. However, the interaction of vimentin filaments with CAS has not been well elucidated. In the present study, stimulation of tracheal smooth muscle strips with acetylcholine (ACh) resulted in the increase in ratios of soluble vimentin to insoluble vimentin (an index of vimentin disassembly) in association with force development. Activation with ACh also induced vimentin phosphorylation at Ser-56 as assessed by immunoblot analysis. More importantly, CAS was found in the cytoskeletal vimentin fraction, and the amount of CAS in cytoskeletal vimentin was reduced in smooth muscle strips upon contractile stimulation. CAS redistributed from the myoplasm to the periphery during ACh activation of smooth muscle cells. The decrease in distribution of CAS in cytoskeletal vimentin elicited by ACh was attenuated by the downregulation of p21-activated kinase (PAK) 1 with antisense oligodeoxynucleotides. Vimentin phosphorylation at this residue, the ratio of soluble vimentin to insoluble vimentin, and active force in smooth muscle strips induced by ACh were also reduced in PAK-depleted tissues. These results suggest that PAK may regulate CAS release from the vimentin intermediate filaments by mediating vimentin phosphorylation at Ser-56 and the transition of cytoskeletal vimentin to soluble vimentin. The PAK-mediated the dissociation of CAS from the vimentin network may participate in the cellular processes that affect active force development during acetylcholine activation of tracheal smooth muscle tissues.