BACKGROUND: The aim of this study was to evaluate the effect of 4-aminopyridine (4-AP) combined with anticholiesterase (antiChE) in antagonizing MgSO4-rocuronium-induced neuromuscualr blockade using a rat hemidiaphragm. METHODS: A hemidiaphragm with phrenic nerve was dissected and was mounted in a bath containing oxygenated Krebs solution. The phrenic nerve was stimulated supramaximally and the twitch response (0.1 Hz) was stabilized for at least 30 minutes. After maximal twitch inhibition by IC95 (concentration of 95% twitch inhibition) of rocuronium and MgSO4 20 mg was achieved, antagonistic effects of 1.6, 16 microgram/ml of edrophonium, 0.1, 1.0 microgram/ml of neostigmine, 0.5, 5.0 microgram/ml of pyridostigmine, and 0.8 microgram/ml of 4-AP combined with each of the above mentioned antiChEs were investigated. RESULTS: Whereas antiChE alone at low concentration partially recovered only the twitch response, 4-AP combined with antiChE recovered both the twitch and train-of-four responses significantly. CONCLUSIONS: 4-AP enhances antagonism of a magnesium-rocuronium induced neuromuscular blockade by edrophonium, neostigmine or pyridostigmine in vitro.