The mitochondrial precursor protein horse heart apocytochrome c was spin-labeled on the cysteine residue at position 14 or 17 in the N-terminal region, and the mature protein yeast cytochrome c was similarly labeled on the single free cysteine residue at position 102 at the C-terminal. The proteins were bound to negatively charged phospholipid bilayers, and the accessibility of the spin-labeled cysteine residues to lipid-soluble molecular oxygen and to the lipid-impermeant chromium oxalate anion was determined from the saturation properties of the ESR spectra. Binding of the protein was found to have a considerable effect on the local oxygen concentrations within the lipid bilayer. The accessibilities of the spin-labeled proteins relative to those obtained for phospholipids spin-labeled either in the headgroup or at positions in the sn-2 acyl chain, in the presence of unlabeled protein, identify the position of the spin-labeled cysteine residues in the phospholipid bilayer. The spin label on apocytochrome c bound to phosphatidylglycerol bilayers lies between the 5- and 14-C positions of the lipid acyl chain. Admixture of > or = 75 mol % phosphatidylcholine induces an additional surface-associated apocytochrome c population. The spin label on native and heat-denatured cytochrome c is located at the membrane surface. These different extents of membrane penetration correlate also with the reduction in local oxygen concentration experienced by spin-labeled phospholipids on binding of apo- and holocytochrome c. The possible biological implications of the data are discussed.