The alpha-lactalbumin segment which penetrates into phosphatidylserine/phosphatidylethanolamine vesicle bilayer under acidic condition was photoactively labeled with 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which had been partitioned into the hydrophobic interior of the bilayer. The hydrophobically labeled amino acid residues were identified by trypsin digestion of the alpha-lactalbumin/vesicle complex, extraction and Edman degradation of the membrane embedded fragment. The results are consistent with a notion that the segment exists in the membrane as an alpha-helix and that only one surface of this alpha-helix is exposed to the hydrophobic interior of the bilayer. Possible models are: (a) a loop of tightly held alpha-helix penetrating deep into the bilayer and (b) the helix being located on the interface between bilayer and the aqueous solution. The time-dependent [125I]TID labeling process revealed that the middle part of this segment goes into the bilayer first and is then followed by both ends. The penetration rate is comparable to that of the fusion of the lipid vesicles of the same composition by alpha-lactalbumin at the same pH, which further supports that the penetration is the cause of fusion.