- Abstract The binding of hematoporphyrin derivative to membrane was studied at the molecular level, employing fluorimetric techniques and using liposomes (large unilamellar) to model biological membranes. Two specific issues were probed: (a) the effect of increasing the porphyrin-liposome incubation period (37°C, neutral pH, in the dark) from 2 h up to 24 h, with liposomes composed of PC or PC / cholesterol 7:3 (molar ratio); (b) the effects of membrane lipid composition, in terms of mol% cholesterol in PC / cholesterol liposomes, on the porphyrin-membrane binding equilibrium, for a long incubation period (16 h). The data were processed and found to be in good agreement with the following proposed model: with time ( t > 2 h), the porphyrin fractions into three components, two of them binding to membrane with high and low affinities, respectively, the time effect reaching a plateau at 16 h. Linkage was observed between the slow process and changes in the available pools of each fraction. At sufficiently long incubations, the magnitude of each of the two binding the magnitude of the binding constant was found to depend on the lipid composition, that of the high-affinity the magnitude of the binding constant was found to depend on the lipid composition, On the other hand, fractions was found to be (roughly) 30%, independent of the membrane lipid composition. On the other Hand, fraction decreasing from 9000 M −1 for 0% cholesterol, to 3000 M −1 for 40% cholesterol, then increasing back to 5000 M −1 upon further increase in cholesterol (to 50% mol) and that of the low-affinity fraction going from 1000 M −1 , to 100 M −1 , to 300 M −1 for similar lipid compositions. The origin of the time effect, in terms of porphyrin-specific processes, and the biological relevance of the present findings are discussed.