Abbreviations used: BSM -bovine brain sphingomyelin; DMPCdimyristoylphosphatidylcholine; DPPC -dipalmitoylphosphatidylcholine; EPR -electron paramagnetic resonance; ESM -egg sphingomyelin; FOT -fast oxygen transport; l d -liquid-disordered; l o -liquid-ordered; PC -phosphatidylcholine; PSMpalmitoylsphingomyelin; 5-SASL -5-doxylstearic acid spin label; SLOT -slow oxygen transport; s o -solid-ordered; T 1 -spin-lattice relaxation time Abstract: Membranes made from binary mixtures of egg sphingomyelin (ESM) and cholesterol were investigated using conventional and saturation-recovery EPR observations of the 5-doxylstearic acid spin label (5-SASL). The effects of cholesterol on membrane order and the oxygen transport parameter (bimolecular collision rate of molecular oxygen with the nitroxide spin label) were monitored at the depth of the fifth carbon in fluid-and gel-phase ESM membranes. The saturation-recovery EPR discrimination by oxygen transport (DOT) method allowed the discrimination of the liquid-ordered (l o ), liquid-disordered (l d ), and solid-ordered (s o ) phases because the bimolecular collision rates of the molecular oxygen with the nitroxide spin label differ in these phases. Additionally, oxygen collision rates (the oxygen transport parameter) were obtained in coexisting phases without the need for their separation, which provides information about the internal dynamics of each phase. The addition of cholesterol causes a dramatic decrease in the oxygen transport parameter around the nitroxide moiety of 5-SASL in the l o phase, which at 50 mol% cholesterol becomes ~5 times smaller than in the pure ESM membrane in the l d phase, and ~2 times