Studies on the membrane binding of cytochrome c revealed liposome-associated and soluble cytochrome c not to be in rapid equilibrium. In brief, cytochrome c attached to pyrene phospholipid-labeled, fluorescent liposomes containing either 17.6 mol % cardiolipin (CL) or 30 mol % egg phosphatidylglycerol (PG) is practically not at all or very slowly, respectively, detached by a subsequently added excess (up to 20-fold) of nonlabeled liposomes containing these acidic lipids. Cytochrome c was fully dissociated from PG-containing liposomes by increasing the ionic strength by NaCl, whereas dissociation from CL-containing membranes was less complete, presumably because of the scavenging of the protein within inverted intramembrane micelles. Importantly, the apparent irreversibility of the binding of cytochrome c to liposomes is strongly dependent on the structure of the acidic phospholipid. Cytochrome c bound to lyso-PG/PC liposomes could be dissociated with an excess of nonlabeled PG-containing liposomes. Cytochrome c was also efficiently bound to membranes containing the negatively charged dicetylphosphate yet could be readily dissociated by nonlabeled PG-containing liposomes. We conclude both proper geometry of the phosphate group and the presence of two acyl chains to be required for the tight binding of cytochrome c to acidic phospholipids. These data provide evidence for the membrane association of cytochrome c by an acidic phospholipid in the extended conformation (Kinnunen, P. K. J., Köiv, A., Lehtonen, J. Y. A., Rytömaa, M., and Mustonen, P. (1994) Chem. Phys. Lipids 73, 181-207) in which one of the acyl chains of the lipid becomes accommodated within a hydrophobic cavity of the protein. Based on the crystal structure of cytochrome c we putatively assign the invariant Asn-52 (horse heart cytochrome c) as the site liganding the protonated phosphate of the lipid, whereas Lys-72 and -73 should bind the deprotonated form.