1. The mechanism of action of the muscle paralysing property of the antibiotic spectinomycin has been investigated on the isolated mouse phrenic nerve-hemidiaphragm preparation. 2. Spectinomycin produced a neuromuscular blockade that was poorly reversed by neostigmine (3 mumol/l) but well reversed by both calcium (5 mmol/l) and 3,4-diaminopyridine (0.1 mmol/l). 3. Intracellular techniques were used to record endplate potentials and miniature endplate potentials from hemidiaphragms paralysed by spectinomycin. Miniature endplate potentials were reduced both in frequency and in amplitude. The quantal content of the endplate potential during spectinomycin paralysis calculated by the method of variance was 14, a value similar to that found in preparations paralysed by the prejunctionally active magnesium. 4. Thus spectinomycin possesses weak neuromuscular blocking activity by a predominant action on acetylcholine release.