Killer toxin from Saccharomyces cerevisiae inhibited the pumping of protons into the medium by metabolically active sensitive cells. Such inhibition coincided with that of the uptake of potassium ions which are thought to be accumulated by yeast cells in order to neutralize the membrane potential created because of the extrusion of protons. The consumption of glucose, however, was identical in killer-treated and untreated cells. These alterations can be explained by the ability of the toxin to reduce the chemical proton gradient across the plasma membrane as measured by the accumulation of the weak permeable [14C]propionic acid. With this method, an internal pH of 6.42 was calculated from normal cells (the external pH was 4.6) while that of toxin-treated cells was decreased as a function of time. The proton concentration gradient was reduced from 66- to 17-fold. It is shown that the toxin-induced alteration of the proton gradient is due to an enhanced proton permeability of the yeast plasma membrane upon binding of the toxin. It is suggested that killer toxin acts as a macromolecular proton conductor similar in some respects to the known proton conductors 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, since all the described effects are also observed with these substances.