The diuretic amiloride has been suggested as a specific inhibitor of T-type neuronal Ca2+ channels. The effects of amiloride on glutamate receptor-gated cationic channels and glutamate-induced. Ca2(+)-dependent neuronal death were investigated in primary neuronal cultures from neonatal rats. In primary cultures of cerebellar granule neurons of the rat, receiving 50 microM glutamate for 15 min, at 22 degrees C, in the absence of Mg2+, about 80% of neurons were killed in about 24 hr. Exposure of neurons to such a pulse of glutamate, in the presence of various concentrations of amiloride, resulted in a dose-dependent protection from neurotoxicity (EC50 300 microM, complete protection 1 mM). In voltage-clamped cortical and cerebellar neurons of neonatal rats in primary culture, 100 microM amiloride diminished (by about 25%) glutamate- and/or NMDA-evoked cationic currents, recorded in the whole-cell mode. About 80% of the NMDA-(20 microM) stimulated current was inhibited by 700 microM amiloride. The inhibitory effect of amiloride was not voltage-dependent. In outside-out membrane patches, excised from granule cells and held at -50 mV, 100 microM amiloride changed the NMDA-elicited single channel activity into a fast flickering between the open and closed states. The noise analysis of the data revealed that, although resembling the Mg2(+)-induced flickering, the amiloride-induced channel block was more similar to the effects described for the action of local anaesthetics on the nicotinic cholinergic channel. The pharmacological relevance of this action of amiloride requires further characterization; the data point out the necessity of a cautious use of amiloride in studying neuronal function.