We purified a new type of polyamine neurotoxins called spidamine and joramine in the venom of a spider, Nephila clavata, on a large-scale and confirmed their structures by chemical analysis and NMR spectra. Spidamine was N-(2,4-dihydroxyphenylacetyl-L-asparaginyl)-N'-(3-aminopropyl-B-alanyl)-1,5-pentanediamine, whereas joramine was N-(4-hydroxyphenylacetyl-L-asparaginyl)-N'-(3-aminopropyl-β-alanyl)-1,5-pentanediamine.We also studied the toxins biological activity on glutamatergic transmission using lobster neuromuscular synapses. The blocking activity of both toxins on the excitatory post synaptic potentials (EPSPs) in the lobster muscle was about one tenth time less than JSTX-3, the most potent toxin in the venom of Nephila clavata. Interestingly, spidamine which has 2,4-dihydroxyphenyl group showed an irreversible block of EPSPs, whereas joramine possessing 4-hydroxyphenyl group had a reversible block.