Joro spider toxin (JSTX), a specific blocker of glutamate receptors, was found to exert a prominent suppressive action on the Na+-dependent binding of L-glutamate to synaptic membranes and on glutamate uptake by synaptosomes in a dose-dependent manner. In contrast, the synthesized 2,4-dihydroxyphenylacetylasparagine (2,4-DHPA-ASN), a common moiety of spider toxins, which has been shown to exhibit almost the same activity as intact JSTX with respect to the inhibition of Na+-independent glutamate binding to its synaptic membrane receptors, shows lower potency in inhibiting Na+-dependent binding and uptake of L-glutamate. From these findings, it is clear that JSTX has the ability to inhibit not only L-glutamate binding to its synaptic membrane receptors but also L-glutamate uptake by synaptosomes, and that polyamines linked to 2,4-DHPA-ASN in the molecule of spider toxins may participate in the inhibition of L-glutamate uptake.