The staphylococcal α‐hemolysin (αHL) and leukocidin (Luk) polypeptides are members of a family of related β‐barrel pore‐forming toxins. Upon binding to susceptible cells, αHL forms water‐filled homoheptameric transmembrane pores. By contrast, Luk pores are formed by two classes of subunit, F and S, rendering a heptameric structure displeasing on symmetry grounds at least. Both the subunit stoichiometry and arrangement within the Luk pore have been contentious issues. Here we use chemical and genetic approaches to show that (1) the predominant, or perhaps the only, form of the Luk pore is an octamer; (2) the subunit stoichiometry is 1:1; and (3) the subunits are arranged in an alternating fashion about a central axis of symmetry, at least when a fused LukS‐LukF construct is used. The experimental approaches we have used also open up new avenues for engineering the arrangement of the subunits of β‐barrel pore‐forming toxins.