The structure-activity relationship of phenylene-polymethylene bis-ammonium (PMBA) derivatives, C6H4[X(CH2)nR]2, on isolated mouse phrenic nerve-diaphragm muscle was investigated to obtain more potent and stable compounds for use as pharmacological tools to clarify the mechanism of succinylcholine (SuCh)-induced neuromuscular blockade. The neuromuscular blocking effect of all the PMBA derivatives was not reversed by neostigmine, a cholinesterase inhibitor. The potency of the neuromuscular blockade was in the order p- > o- > m- with respect to the side-chain substituents. A PMBA composed of X = CH2, n = 5 and R = N+Et3 was 5.9- and 23-fold more potent than SuCh and decamethonium, respectively. The derivatives of R = N+Et3 were observed to be more potent than those of R = N+Me3, N-Me-piperidinio and pyridinio derivatives. Replacement of X = CH2 with O, CHOH and CHOAc decreased the neuromuscular activity while replacement with S, SO and SO2 increased it. Introduction of NO2 into the phenylene ring increased the activity, while the introduction of an alcohol, aldehyde and ketone group decreased it. Removal of a carbonyl or ether group from SuCh decreased its activity, whereas the introduction of these into PMBA failed to increase it. We managed to synthesize unhydrolyzable neuromuscular blocking agents which are more potent than SuCh.