Membrane±water partitioning of inhibitors of acyl-coenzyme A:cholesterol acyltransferase (ACAT) governs the concentration of inhibitor that ACAT is exposed to and determines the corresponding extent of cholesterol esteri®cation inhibition. Partitioning of the ACAT inhibitors CI-976, CL 277,082, and SaH 58-035 into rat liver microsomes containing ACAT was detected by shifts in the level of inhibition that were independent of inhibitor concentration but inversely dependent on microsome membrane concentration. The equilibrium distribution of the ACAT inhibitors between aqueous and membrane phases was derived directly from these data by application of a previously described method of linear analysis. The accuracy of membrane partitioning analysis based on kinetic data was veri®ed for CI-976 by direct measurements of [ 14 C]CI-976 partitioning into phospholipid membranes. The results show that the ACAT inhibitors are highly partitioned into membranes by factors exceeding 1 Â 10 6 . This result is consistent with the far greater in¯uence of membrane content over aqueous volume on inhibitor activity. The results demonstrate that the size of the membrane phase in aqueous suspension must be taken into account to obtain accurate and reproducible kinetic characterizations of membrane-active molecules. Analyses of the membrane-dependent shifts in activity can be used to calculate the membrane±water partitioning coef®cient of membrane-active molecules such as ACAT inhibitors.