Microsomal phosphatidate phosphohydrolase (phosphatidate phosphatase EC 184.108.40.206) was solubilized and fractionated to yield at least two distinct enzymatically active fractions. One, denoted FA, was non-specific, had a relatively high Km for phosphatidic acid and was insensitive to inhibition by diacylglycerol. The second fraction, FB, was specific for phosphatidates, had a low Km, and was inhibited, non-competitively, by diacylglycerol. FA exhibited a sigmoid substrate-activity curve. The isolated FB aggregated to particles of about 10(6) in the absence of salts and could be dissociated by the addition of monovalent cations at ionic strength 0.4-0.6 to about 2-10(5) daltons and thereby doubled its activity. Dissociation was time- and temperature-dependent. F- was inhibitory. Divalent ions were not required for the activity of FA or FB and inhibited at concentrations exceeding 1 mM.