The interaction of d-tubocurarine, pancuronium, or succinylcholine with neomycin, streptomycin, or polymyxin B was investigated using a rat phrenic nerve-hemidiaphragm preparation. All neuromuscular blocking agents (relaxants) mutually potentiated the neuromuscular blocking action of one another; combinations of ineffective concentrations of relaxants and antibiotics caused an 82 to 98% neuromuscular block. This extensive potentiation of the neuromuscular effects of relaxants by antibiotics can be attributed to the fact that antibiotics not only have a curare-like stabilizing effect on the post junctional membrane, but also decrease presynaptic acetylcholine release. Neostigmine (0.25 μg/ml) only partially antagonized the neuromuscular block caused by the various drug combinations. In contrast, 4 7mu;g/ml of 4-aminopyridine returned the twitch tension, depressed by combined administration of relaxants and antibiotics, to or above control values except in the case of neuromuscular block caused by the combinations of succinylcholine and polymyxin B.