Aerolysin is a bacterial toxin that binds to a receptor on eukaryotic cells and oligomerizes to form stable, SDS-resistant, noncovalent oligomers that insert into the plasma membrane and produce well-defined channels. Little is known about the mechanisms controlling this process. Here we show that the protonation of a single histidine is required for oligomerization of aerolysin in solution. First we have investigated the effect of pH on the activity of aerolysin. The toxin's ability to disrupt human erythrocytes declined as the pH increased above 7.4. Experiments with receptor-free planar lipid bilayers demonstrated that the rate at which aerolysin formed channels also decreased with increasing pH, although the conductance of preexisting channels was not affected. The reduction in the rate of channel formation was shown to be due to a decrease in the toxin's ability to oligomerize. Our data indicate that the pH effect on activity is due to the deprotonation of a single residue rather than a global effect of pH on the protein. In agreement with our previous site-directed mutagenesis studies, His-132 is most likely to be the target of this pH effect. This conclusion was reinforced by the fact that we could shift the pH dependence of the activity to lower pH values by mutating Asp-139, a residue less than 3 A away from His-132 and likely to contribute to the usually high pKa of this histidine.