Relative partition coefficients of fatty acids and alcohols between aqueous buffers and biological membranes have been determined from the linear relationship between isotope content of sedimented membranes and aqueous concentration. This technique allows study of highly lipid soluble compounds such as long-chain saturated fatty acids. Rat intestinal brush border membranes and erythrocyte ghost membranes were studied by using homologous series of saturated fatty acids, mono-unsaturated fatty acids and 10, 12, and 14 carbon normal alcohols. The influence of chain length on partitioning was similar in the three series with an incremental free energy of -820 cal/mole per methylene group in brush borders for the saturated fatty acids. Incremental enthalpy and entropy were -1331 cal/mole and -1.64 cal/mole, degrees K respectively. Decrease in the partition coefficient due to the double bond (monounsaturated relative to saturated) had an incremental free energy of +1178 cal/mole, incremental enthalpy of -3453 cal/mole, and incremental entropy of -7.34 cal/mole, degrees K, while substitution of the hydroxyl for the ionized carboxyl group (pH 7.4) increased the partition coefficient by 72-fold. From these data it must be concluded that the lipid phase of the membrane bilayer is extremely hydrophobic, similar to heptane or polyethylene in polarity.