Oxygen transport in phosphatidylcholine-cholesterol membranes has been studied by observing the collision of molecular oxygen with nitroxide radical spin labels placed at various distances from the membrane surface using long-pulse saturation recovery ESR techniques. The collision rate was estimated for tempocholine phosphatidic acid ester, 5-doxylstearic acid, and 16-doxylstearic acid from spin-lattice relaxation times (T1) measured in the presence and absence of molecular oxygen. Profiles of the local oxygen transport parameter across the membrane were obtained as a function of cholesterol mol fraction and temperature in L-alpha-dimyristoylphosphatidylcholine ([ Myr2]PtdCho) and L-alpha-dioleoylphosphatidylcholine ([ Ole2]PtdCho) membranes. Membrane oxygen permeability coefficients were estimated from oxygen transport parameter profiles. At approximately 30 degrees C, the oxygen permeability coefficients in the presence and absence of 50 mol % cholesterol are 22.7 and 125.2 cm/s, respectively, for [Myr2]PtdCho membranes, and 54.7 and 114.2 cm/s, respectively, for [Ole2]PtdCho membranes (compared with 60-80 cm/s for water layers with the same thicknesses as the membranes). The major results in the liquid-crystalline phase are as follows: (i) In the absence of cholesterol, membranes are not barriers to oxygen transport. (ii) Addition of 50 mol % cholesterol decreases oxygen permeability by a factor of approximately 5 and approximately 2.5 in [Myr2]PtdCho and [Ole2]PtdCho membranes, respectively. The resistance to oxygen transport is located in and near the polar headgroup regions in the membrane. (iii) Cholesterol increases oxygen transport in the central regions of [Ole2]PtdCho membranes.