[structure: see text] 5-(Cholesteryloxyethoxyphosphoryl)-5-methylpyrroline N-oxide (5-ChEPMPO), a DEPMPO analogue bearing a cholesterol group on the phosphorus atom, has been prepared and used to trap peroxyl-, alkoxyl-, thiyl-, and carbon-centered radicals in organic solvent. The important steric hindrance in 5-ChEPMPO does not affect the properties of 5-ChEPMPO in comparison to DEPMPO for the spin trapping of an enantiopure linoleic acid hydroperoxide. The 5-ChEPMPO-OOL spin adduct was observed by ESR and confirmed by ESI-MS/MS experiments. The relaxation terms of the 5-ChEPMPO-lipid peroxyl spin adduct were compared with those of other peroxyl spin adducts, and it was shown that the cholesteryl group has only a weak influence on the exchange rate between adduct conformers.