1. Using whole-cell patch-clamp techniques, the mechanisms of NMDA channel blockade by amino-adamantane derivatives (AADs) memantine (3, 5-dimethyl-aminoadamantane, MEM) and amantadine (1-aminoadamantane, AM) have been studied in rat hippocampal neurons acutely isolated by the vibrodissociation method. A rapid concentration-jump technique was used to replace superfusing solutions. 2. The aspartate (Asp)-induced channel opening greatly accelerated but was not a prerequisite for the recovery from the block by MEM: it was able to leave the channel without agonist assistance. The co-agonist (glycine) as well as the competitive NMDA antagonist DL-2-amino-7-phosphonoheptanoic acid (APV), did not affect this recovery. Membrane depolarization accelerated it, strongly suggesting that this process proceeded via the hydrophilic pathway of the channel. 3. A comparison of the kinetics of the recovery from the block by AADs in the presence and absence of the agonist prompted a hypothesis that the blocker trapped in the channel increased the probability of its transition to the open state. 4. Both MEM and AM were able to block NMDA channels not only in the presence but also in the absence of Asp, although in the latter case the effective blocking concentrations were much higher and the rate of the block development was much smaller than in the former case. The extent of the block increased with the duration of the blocker application. Glycine enhanced this block, while APV attenuated it. The MEM-induced blockade of agonist-unbound channels was enhanced by membrane hyperpolarization and weakened by external Mg2+. These findings strongly suggested that the blocker reached its binding sites via the same hydrophilic pathway both in the presence and absence of the agonist. 5. A comparative analysis of the channel unblocking kinetics in the presence of Asp after their blockade with or without the agonist assistance led us to conclude that in the two cases AADs were bound to the same blocking sites in the channel.