Both the Ca2+ signal and the alteration of the Ca2+ sensitivity of the contractile apparatus regulate smooth muscle contraction. Myosin light chain kinase (MLCK) phosphorylated the 20 kDa regulatory myosin light chain (MLC20) resulting in contraction. Myosin light chain phosphatase (MLCP) dephosphorylates MLC20 causing relaxation. Thus, the balance between the activities of MLCK and MLCP determines the level of MLC20 phosphorylation.MLCP consists of a 38 kDa catalytic subunit (PP1cδ), a 110 kDa targeting subunit (MYPT1), and a 21 kDa small subunit (M21). MYPT1 provides the substrate specificity and the regulation of phosphatase activity. It was reported that myosin phosphatase-Rho interacting protein (M-RIP) bound MYPT1 and thus targeted MLCP to the actomyosin contractile filament based on yeast-two hybrid and cell biological assays.To determine if MYPT1 binds to M-RIP directly, we performed analytical ultracentrifugation (AUC) study using purified peptides of MYPT1 and M-RIP. Circular dichroism and AUC analysis illustrated that the M-RIP peptide spanning residues 724-878 of M-RIP is a coiled coil and forms a dimer. The AUC analysis demonstrated that the C-terminal coiled-coil region of MYPT1 spanning residues 924-991 did not bind the M-RIP peptide, whereas the C-terminal random coiled-coil region of MYPT1 (synthetic LZ) spanning residues 991-1030 did bind, forming a heterotrimer. In addition, three individual glutamic acid residues (amino acids 998-1000) of MYPT1 were critical for binding. We replaced the glutamic acids either all three at a time or one at a time with glutamine residues. In addition, we replaced all three glutamic acids with aspartic acids. However none of these mutants bound to synthetic LZ demonstrating that these three glutamic acid residues are essential for binding.