The lipid membrane partitioning of lysolipids (lysoPC) and fatty acids (FA) into unilamellar vesicles composed of saturated DC16PC phospholipids has been determined by means of isothermal titration calorimetry (ITC). The calorimetric titrations were performed at low temperatures in the ordered gel phase and at high temperatures in the disordered fluid phase of the phospholipid membrane vesicles. The long saturated acyl chains of the lysolipids and fatty acids varied from 10 to 16 carbon atoms and all titrations were performed below the critical micellar concentrations (cmc) of the detergents. The calorimetric results reveal that the membrane partitioning of lysolipids depends strongly on the phase structure of the lipid membrane. This is manifested as a lysolipid partition coefficient, K, that is much larger for fluid-phase lipid membranes as compared to gel-phase lipid membranes. Oppositely, the membrane partitioning of fatty acids depends only weakly on the phase structure of the phospholipid vesicles. In addition, the thermodynamic measurements show that the partition coefficients for both the lysolipids and fatty acids toward gel and fluid lipid membranes become almost an order of magnitude higher when the saturated acyl chain of the detergents increases by two carbon atoms. The obtained partition coefficients are of importance in relation to a deeper understanding of the interplay between global aqueous and local membrane concentrations of the detergents and the functional influence on, for example, the lipid membrane permeability and the activity of membrane associated enzymes such as phospholipase A(2).