Recent pharmacological studies have led to the hypothesis that aminoglycoside-induced ototoxicity is an excitotoxic process mediated, at least in part, by a polyamine-like modulation of N-methyl-D-aspartate (NMDA) receptors. To explore this hypothesis, we compared the effects of several aminoglycosides (neomycin B, kanamycin A, streptomycin, and dihydrostreptomycin) with spermine on recombinant NMDA receptors of defined composition expressed in Xenopus oocytes. Like spermine, aminoglycosides potentiate agonist-induced responses in the presence of both saturating glycine ("glycine-independent") and subsaturating glycine ("glycine-dependent") concentrations on NR1A/2B receptors. Likewise, aminoglycosides and spermine potentiated the agonist responses under glycine-dependent conditions on NR1A/2A receptors. Despite these similarities, several prominent differences were observed between spermine and aminoglycosides as well as among individual aminoglycosides. For example, neomycin B, streptomycin, and kanamycin A, but neither spermine nor dihydrostreptomycin, potentiated glycine-dependent responses on NR1A/2C receptors. Differences between spermine and aminoglycosides also were observed with respect to the voltage dependence of polyamine-like actions. For example, under glycine-dependent conditions, potentiation at NR1A/2B receptors by spermine was voltage dependent, decreasing as the holding potential was changed from -35 to -85 mV; in contrast, potentiation induced by aminoglycosides at NR1A/2B receptors was voltage independent. No direct relationships emerged between the effect of an aminoglycoside at a specific NMDA receptor subtype and the ototoxicity of these antibiotics.