Phosphatidate phosphohydrolase (PAP) from cytosolic fraction of rat liver was purified to homogeneity having specific activity of 5.14 U/mg protein. An activity staining procedure was developed to determine molecular weight of the enzyme on polyacrylamide gel electrophoresis using Ferguson plot. Molecular Weight (M.W.) of the active PAP was 298 KDa. SDS-PAGE analysis showed a M.W. of 47 KDa for PAP subunits. Active multimer of the enzyme, therefore, was calculated to be hexamer. Gel filtration on Sephadex G-100 column showed a M.W. of 850 KDa for PAP due to the protein aggregation on the matrix. The purified enzyme was inhibited by divalent cations such as Fe2+, Cu2+ and Ca2+ but requires Mg 2+ for its activity. The activity loss of PAP inhibited by cations was restored by Mg2+ on polyacrylamide gel. The data suggest that the active form of cytosolic PAP is a hexamer of identical subunits and that charge density plays an important role in enzyme-substrate interaction. Magnesium ion is probably the only divalent cation capable of generating proper enzyme-metal-substrate complex necessary for the catalytic activity.