Contractile processes in non-muscle cells mimic those occurring in smooth muscle tissues, indicating the value of using non-muscle cells to investigate the biochemical pathways that regulate contraction (4 -8). Phosphorylation of myosin light chains (MLC) in non-muscle cells causes the formation of myosin-containing stress fibers, which are contractile bundles of actin filaments associated with myosin II (4 -8). Phosphorylation of MLC similarly increases actin-myosin interactions in smooth muscle cells, resulting in smooth muscle contraction (reviewed in Refs. 9 -12). In addition to smooth muscle contraction, many fundamental cellular processes such as adhesion, migration, and division depend upon the interaction of myosin with actin in contractile filaments (reviewed in Ref. 12). Activation of mAChR may affect these fundamental processes by altering myosin activity in non-muscle cells as it does in smooth muscle. Although mAChR activation induces MLC phosphorylation and subsequent contraction in smooth muscle cells (13)(14)(15)(16)(17), the ability of mAChR to regulate MLC phosphorylation and myosin organization in non-muscle cells has not been reported.We investigated the ability of transfected human mAChR subtypes to regulate myosin organization in Chinese hamster ovary (CHO) cells. Activation of transfected M 3 mAChR induces MLC phosphorylation and causes myosin-containing stress fibers to form in CHO cells. The involvement of PKC in these events is indicated by our findings that 1) direct activation of PKC with phorbol esters induces MLC phosphorylation and myosin reorganization in CHO cells, 2) specific PKC antagonists inhibit M 3 mAChR-mediated myosin reorganization, and 3) activation of transfected M 1 but not M 2 mAChR subtypes also induces the formation of myosin-containing stress fibers, demonstrating that only mAChR subtypes that stimulate PKC activity induce myosin reorganization. The participation of myosin light chain kinase (MLCK) and RhoA in mAChRmediated myosin reorganization was also investigated, since these proteins regulate contractile processes in other systems

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