Gel filtration of rat liver cytosol on Bio-Gel A-5m resolved the phosphatase activities into four peaks, all of which showed activity with either phosphatidase bound to microsomal membrane (PAmb) or phosphatidate dispersed in sonicated microsomal lipid (PAaq) as the substrate. A major part of the PAmb phosphatase activity (52%) was eluted in a peak with an apparent molecular weight (Mr) of 500,000 where the PAaq phosphatase activity was very low. A major PAaq phosphatase activity peak (48%) was obtained in the void volume (Vo), where the PAaq phosphatase activity was higher than the PAmb phosphatase activity. The addition of 0.075% Tween 20 to the elution buffer gave only the 500 kilodalton (kDa) peak. When the activity in the Vo peak obtained in the absence of Tween 20 was rechromatographed in the presence of the detergent, a part of the activity was dissociated into 500 kDa molecules having a preference for PAmb. These results suggest that the enzymes obtained in the Vo peak are formed by the association of the 500 kDa molecules with macromolecules and that the substrate preference of phosphatidate phosphatase is modified by the change in the physical state of the enzyme. The microsomal phosphatidate phosphatase activities were also separated on Bio-Gel A-5m after solubilizing by sonication. Most of both the PAmb and PAaq phosphatase activities were coeluted in the Vo peak, in which the latter activity was higher than the former. When the gel filtration was performed in the presence of Tween 20, a major activity peak with a preference for PAmb was obtained at the elution volume corresponding to apparent Mr 500,000, indicating a potential relationship between the cytosolic and microsomal activities.