The transverse location of trans-parinaric acid in spherical vesicles made up from dipalmitoylphosphatidylcholine has been investigated by the differential quenching of the probe fluorescence by 5- and 16-doxylstearic acid derivatives. The quenching data are interpreted in terms of a local fluorophore concentration factor. In this way it was found that the polyene of t-PnA is located within the inner part of the bilayer (presumably aligned with the bilayer lipids), both in the gel and in the liquid crystalline phases.