A20668 A, B, and C are polypeptide antibiotics that inhibit phosphorylation of ADP, Mg2t-ATPase, and the ATP-driven transhydrogenase of rat liver submitochondrial particles, but not the purified F1 ATPase. In intact mitochondria, 120668 inhibits uncoupler-induced ATPase, State 3 respiration, and phosphorylation; the A and B forms are approximately equipotent with rutamycin, whereas A20668 C is less effective. Concentrations of A20668 slightly greater than required for complete inhibition of phosphoryl transfer stimulate rapid, uncoupled respiration by mitochondria under State 3 of 4 conditions. A20668 A and B are more effective uncouplers than A20668 C. In the presence of venturicidin or ossamycin, concentrations of A20668, which alone do not uncouple, stimulate oxygen consumption of mitochondria incubated under either State 3 of 4 conditions. A20668 uncoupling is not potentiated by prior inhibition of phosphoryl transfer by venturicidin X, rutamycin, aurovertin, or efrapeptin. A20668 increases mitochondrial permeability to protons in passive swelling experiments where facilitation of proton conductance correlates well with potency to uncouple. A20668 apparently binds initially at a unique locus to inhibit mitochondrial phosphoryl transfer reactions. When this site is saturated, additional antibiotic may uncouple by increasing proton conductance of mitochondria. Binding of venturicidin or ossamycin appears to interfere with the binding of A20668 to its adjacent inhibitory site, thus effectively increasing the concentration of A20668 available to uncouple.