An endogenous inhibitor of Ca2+-activated neutral protease (CANP) was purified to homogeneity from the soluble fraction of human platelets by the combination of heat treatment, ammonium sulfate fractionation, ion exchange chromatography and gel filtration. The purified inhibitor was found to be a tetramer composed of identical subunits and each subunit has a molecular weight of 63 K. The purified protein exerted specific inhibition against the low Ca2+-requiring form of CANP (mu-CANP) purified from human platelets in the presence of micromolar concentration of Ca2+. The kinetic study revealed that the inhibition is non-competitive with Ki value of 3.2 X 10(-8) M.