This paper reports the effects of microcin J25 (MccJ25) on the microviscosity and permeability of phospholipid vesicles of different compositions. The results obtained indicate that MccJ25 interacts with egg L-alpha-phosphatidylcholine (PC) vesicles as demonstrated by peptide intrinsic fluorescence determinations. The interaction depends on the lipid composition of the vesicles. MccJ25 interaction induces a significant fluidity increase of egg PC vesicles. This effect is time and concentration dependent. Both trimethyl ammonium 1,6-diphenyl-1,3,5-hexatriene and 1,6-diphenyl-1, 3,5-hexatriene gave the same results. The microviscosity of L-alpha-phosphatidylcholine dipalmitoyl small unilamellar vesicles (SUVs) was affected while that of L-alpha-phosphatidylcholine dimyristoyl vesicles was not, indicating that the effect was strongly dependent on the chain length of fatty acids. On the other hand, negatively charged L-alpha-phosphatidyl-DL-glycerol (PG) vesicles remarkably inhibited the peptide effect. Nevertheless vesicles composed of L-alpha-phosphatidylethanolamine:PG:cardiolipin (7:2:1), a composition resembling bacterial membrane, were sensitive to the MccJ25 effect. MccJ25 effectively dissipated the valinomycin-induced membrane potential, but induced only a modest leakage (5%) of the trapped Tb(+3)-dipicolinic acid complex. These results indicate that the peptides interact and perturb the bilayer of SUVs. The relationships between this effect and bactericidal action remain to be elucidated.