The injury of neurons may be caused not only by inadequate energy supply but direct actions of glutamate which release and accumulation occur during ischemis and neurodegenerative diseases. K⁺-ATP (K-ATP)-mediated current can exert a protective role and we report a novel effect of stimulation of single channels by glutamate in cultured hippocampal neurons. AMPA and kainate (10 mM) increased their activity several fold within 20 s that was manifested by increase in the open state probability of the channels as well as the appearance of new, previously inactive (sleepy channels). The increased activity was maintained for 1 to 10 min after washing out glutamate. After activation of NMDAR (30 mM) and metabotropic GluR with t-ACPD (0.5 mM) the effects were weaker and showed more delay. The effects of agonists are likely mediated by reactive oxygen species (ROS) known to activate K-ATP channels (Mironov & Langohr, 2005). ROS production is enhanced after glutamate-induced increase of calcium. In line with this scenario the effects of AMPA and kainate were not observed in calcium-free solution. The data thus indicate that glutamate release during ischemia can cause immediate and long-lasting stimulation of K-ATP channels in neurons that would be protective and may counterbalance glutamate-assisted neurodegenerative processes.