Anthrax toxin consists of three proteins, protective antigen, lethal factor and oedema factor. A proteolytically activated 63-kDa fragment of protective antigen binds lethal factor/oedema factor and translocates them into the cytosol. Domain II of protective antigen has been implicated in membrane insertion and channel formation. In the present study, alanine substitutions in 14 consecutive residues of the 2beta1 strand that are highly homologous to the putative membrane interacting segment of Clostridium perfringens iota-b toxin were generated and the effect on the biological activity of protective antigen studied. One of the mutants, Pro260Ala, showed considerably reduced toxicity in combination with lethal factor. The mutant also showed decreased membrane insertion and translocation of lethal factor into the cytosol. The data suggest that Pro260 is important for membrane insertion and translocation by protective antigen.