Tetraethylammonium (TEA) (2.6 x 10(-3) M) potentiated the twitches of the indirectly- or directly-stimulated phrenic nerve diaphragm of the rat at 37 degrees C by prolonging the action potential of the sarcolemma, due to an inhibition of the repolarizing K+ current. With indirect stimulation, TEA caused a use-dependent inhibition of tetanic contractions, induced every 10 min by 10 sec of 50 Hz stimulation, and a post-tetanic depression of the twitches was observed after about 40 min. Recording of the electromyogram (EMG) and compound action potentials of the phrenic nerve, localized the two inhibitory effects to the neuromuscular junction. They were caused by different mechanisms of action. Choline (3.6 x 10(-4) M) antagonized the depression of the twitch but not the use-dependent inhibition. Lowering the temperature to 20 degrees C reduced the depression of the twitch, whereas the use-dependent inhibition was enhanced. The release of transmitter was probably normal during tetanic stimulation; a post-synaptic desensitization of acetylcholine (ACh) receptors caused the inhibition. Microelectrode recordings of endplate potentials supported this conclusion. The depression of the twitch was due to a presynaptic depletion of transmitter. This was confirmed by inducing an additional depletion and depression of the twitch with N-ethyl-maleimide (2.5 x 10(-5) M). Since the depression of the twitch was antagonized by choline, the depletion was probably due to an inhibited uptake of choline into the nerve terminals.