The membranous lipase of rat liver microsomes was used to hydrolyze diacylglycerol (DG), generated within the microsomal membrane by treatment with phospholipase C, in two separate interactions. For an intramembrane enzyme-substrate interaction, the enzyme and DG were present in the same microsomes. For intermembrane interactions, native microsomes of rat liver were used as carriers of the enzyme, while heated and phospholipase C-treated microsomes of rat liver or brain were employed as carriers of the substrate. The v vs S curves of the intermembrane interaction were hyperbolic while those of the intramembrane utilization were parabolic.