Myosin light-chain kinase (MLCK) is a multi-domain protein with kinase and actin-binding domains, among others. Deficiency of MLCK expression in GBaSM-4 vascular smooth muscle cells enhanced cell proliferation rate and shortened cell doubling time. Transient transfection of the MLCK-deficient cells with cDNA constructs of either wild-type MLCK or its mutant lacking the kinase activity reverted the cell proliferation rate to that of wild-type cells, whereas that of MLCK lacking the actin-binding domain maintained cell proliferation at an elevated rate similar to the MLCK-deficient cells. Thus, the actin-binding domain of MLCK seems to play a role in regulating cell proliferation.