The barbiturate amytal (5-ethyl-5-isopentylbarbituric acid) has been shown to inhibit amino acid transport in membrane vesicles from anaerobically grown Escherichia coli. Amytal has no effect on the activity of the enzymes of the nitrate respiration system, nor on electron transfer in this system. However, addition of amytal to the membrane vesicles results in a decrease of the membrane potential from -90 mV to -72 mV, and to a decrease of the pH-gradient of -61 mV to undetectable values. Furthermore, amytal causes an increase in the rate of ferricyanide reduction in liposomes, indicating that amytal increases the proton permeability of phospholipid membranes. These results demonstrate that amytal acts as an uncoupler in membrane vesicles from anaerobically grown E. coli.