The effect of pentobarbital sodium was studied in both rat phrenic nerve-hemidiaphragm and rat sciatic nerve-gastrocnemius preparations. It was found that pentobarbital produced twitch depression leading to a complete neuromuscular blockade. The pentobarbital-induced neuromuscular depression was significantly enhanced by d-tubocurarine, succinylcholine and hemicholinium. In chronically denervated muscle, pentobarbital also suppressed acetylcholine (ACh)-contracture. The mechanism of neuromuscular blockade produced by pentobarbital may be (a) reduction of ACh release from motor nerve ending or (b) decrease sensitivity of motor end-plate to Ach.