The role of myosin phosphorylation by myosin light chain kinase (MLCK) in regulating the invasiveness of metastatic cancer cells was investigated using the Dunning rat prostatic adenocarcinoma cell line, Mat Ly Lu, and in vitro invasion assay. Treatment with MLCK inhibitors resulted in marked reduction of invasiveness, which was principally due to impaired cellular motility, whereas the ability to survive and proliferate, to adhere to matrix, and to secrete gelatinases were minimally affected.