AbstractFluorescence quenching of pyrene derivatives by 2,2,6,6‐tetramethylpiperidinyl‐1‐oxy (TEMPO), 4‐oxo‐2,2,6,6‐tetramethylpiperidinyl‐1‐oxy and 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyl‐1‐oxy has been measured in homogeneous solvents and microheterogeneous systems: cetyltrimethylammonium chloride micelles, large unilamellar vesicles of dioctadecyldimethylammonium chloride and dipalmitoylphosphatidylcholine (DPPC) and rat liver microsomes. The extent of intraaggregate quenching is mostly determined by the quencher incorporation to the micro‐phases. In particular, it is observed that quenching by TEMPO in vesicles is considerably faster when the bilayer is in the liquid crystalline state. This significant increase in quenching rate with the melting of the bilayer is not observed for the other TEMPO derivatives, indicating that the effect of the lipid organization upon the solubility is related to the hydrophobicity of the solute. The data obtained in rat liver microsomes at 37°C show a pattern very similar to that observed in DPPC vesicles in