The characteristics of the interaction of mitochondrial creatine kinase (mt-CK) with phospholipid vesicles are determined. The presence of negatively charged phospholipids is required to obtain a significant binding of mt-CK. The interaction seems to be largely of an electrostatic nature: it increases with increasing amounts of anionic phospholipid in liposomes and decreases when the ionic strength increases or when the pH of the medium is higher than the pIof mt-CK. We have compared the effects of various effectors used to solubilize mt-CK from the mitochondrial membrane on the binding of mt-CK to liposomes: the nucleotide substrates ATP and ADP have no influence,parahydroxymercuribenzoate, a negatively charged organomercurial compound, partially decreases mt-CK binding; and the anticancer agent adriamycin efficiently prevents mt-CK binding. As monitored by the increase in absorbance, mt-CK causes vesicle aggregation. A differential scanning calorimetry study, using dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol vesicles, shows that mt-CK produces a decrease in the enthalpy variation without any change in the position of the calorimetric peak maximum. This suggests a partial disorganization of the phospholipid bilayer upon interaction with mt-CK.