The binding of tritiated Leu-enkephalin to phosphatidylserine and phosphatidylcholine vesicles, both unmodified and modified by the incorporation of free fatty acid, has been studied by steric exclusion chromatography, ultraviolet difference spectroscopy and fluorescence anisotropy. The results obtained tend to confirm that both ionic and hydrophobic interactions are important in the binding phenomena. On the other hand, it seems likely that steric factors play a very limited role in the recognition of the phospholipid by the opioid peptide. Finally, these results confirm the existence of three complexes of different size, as already demonstrated. But, unlike the previously presented results, they stress the importance of the larger of the three complexes formed through binding.