Low pH-induced fusion mediated by the hemagglutinin (HA) of influenza virus involves a conformational change in the protein that leads to the insertion of a "fusion peptide" of the protein into the target membrane. It has been suggested that this insertion, aided by the formation of a complex of multiple HA trimers, would lead to perturbation of the bilayer structure of the membrane, initiating fusion. Here we present data showing that the interaction of the bromelain released ectodomain of the protein (BHA) with liposomal membranes at low pH leads to pore formation, at least at low temperatures. Strongly temperature-dependent low pH-induced inactivation of BHA resulted in a complete lack of activity of BHA above 10 degrees C. Even at 0 degrees C, only about 5% of the BHA participated in pore formation. Viral HA was less rapidly inactivated and still induced pores at 37 degrees C. BHA-induced pore formation showed a sigmoidal time course. Once BHA had formed a pore in one liposome, it did not form a pore in a further liposome. Quantitative analysis of pore formation indicated that one single BHA trimer sufficed to produce a pore. These data indicate that fusion peptide insertion perturbs the membrane and that the formation of a complex of trimers is not a prerequisite for the perturbation.