A peptide with a sequence corresponding to the highly conserved α-5 segment of the Cry δ-endotoxin family (amino acids 193-215 of Bacillus thuringiensis CryIIIA, was investigated with respect to its interaction with insect membranes, cytotoxicity in vitro towards Spodoptera frugiperda (Sf-9) cells, and its propensity to form ion channels in planar lipid membranes (PLMs). Selectively labelled analogues of α-5 at either the N-terminal amino acid or the ε-amine of its lysine, were used to monitor the interaction of the peptides with insect membranes. The fluorescent emission spectra of the 7-nitrobenz-2-oxa-1,3-diazole-4-yl (NBD)-labelled α-5 peptides displayed a blue shift upon binding to insect (Spodoptera littoralis) mid-gut membranes, reflecting the relocation of the fluorescent probes to an environment of increased apolarity, i.e. within the lipidic constituent of the membrane. Moreover, midgut membrane-bound NBD-labelled α-5 peptides were protected from enzymic proteolysis. Functional characterization of α-5 has revealed that it is cytotoxic to Sf-9 insect cells, and that it forms ion channels in PLMs with conductances ranging from 30 to 1000 pS. A proline-substituted analogue of α-5 is less cytolytic and slightly more exposed to enzymic digestion. Molecular modelling utilizing simulated annealing via molecular dynamics suggests that a transbilayer pore may be formed by α-5 monomers that assemble to form a left-handed coiled coil of approximately parallel helices. These findings further support a role for α-5 in the toxic mechanism of δ-endotoxins, and assign α-5 as one of the transmembrane helices which form the toxic pore. The suggested role is consistent with the recent finding that cleavage of CryIVB δ-endotoxin in a loop between α-5 and α-6 is highly important for its larvicidal activity.