It had been suggested that influenza virus-mediated membrane fusion might be dependent on a pH gradient across a target membrane. We have designed experiments in which this issue could be addressed. Two populations of liposomes were prepared, both simulating the plasma membrane of target cells, but with the pH of the internal aqueous medium buffered either at pH 7.4 (physiological cytosol pH) or at pH 5.0 (endosomal pH at which influenza virus displays maximal fusion activity). By monitoring fusion using the R18 assay, we found that the internal pH of the target liposomes did not influence membrane merging as mediated by the influenza virus hemagglutinin, thus demonstrating that a transmembrane pH gradient is not required in this fusion process.