MARCKS(myristoylated alanine-rich C kinase substrate), a PKC substrate, is a filamentous actin binding protein. A hypothesis for the dimerization of MARCKS protein was evaluated. MARCKS mediated filamentous actin cross-linking either by dimerizing MARCKS which bound to filamentous actin or by the direct interaction of MARCKS with two filaments at separate binding sites. MARCKS was purified from rat brain by immunoaffinity chromatography. MARCKS dimerization was examined by chemical cross-linking using glutaraldehyde, disuccinimidyl suberate(DSS), dimethyl suberimidate(DMS) and ethyl-[3-dimethyl aminopropyl]-carbodiimide hydrochloride(EDAC) and by sucrose gradient rate-zonal centrifugation. The results suggested that MARCKS existed as a monomer. Phosphorylation specificities to the subcellular locations of MARCKS were also tested in mouse splenocytes. MARCKS in the membrane fraction was found to be mostly dephosphorylated while MARCKS in the cytosolic fraction was largely phosphorylated. It was found that the treatment of PKC agonist, PMA, increased phosphorylated MARCKS with the subcellular displacement from membrane to cytosol.