Apocytochrome c has a potent ability to insert spontaneously into membrane. To identify which sequences were critical for this insertion activity, a series of peptides N19, C8, C15 and C21, corresponding to sequences 1-19, 81-88, 74-88 and 68-88 of apocytochrome c, respectively, were synthesized and purified. Insertion ability into phospholipid monolayer, intrinsic fluorescence emission spectra, and the accessibility of peptide C21 to fluorescence quenchers: KI, acrylamide and HB showed that only segment 68-88 could insert into membrane, while other segments did not. CD spectra demonstrated that its interaction with liposomes containing negatively charged phospholipid could induce a partial alpha-helical conformation in peptide C21. It is interesting to note that a cooperation exists between segment 68-88 and 1-19 in the insertion of apocytochrome c and consequently translocation across membrane.