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Rat cortical neurons cultured for 3 days in vitro were loaded with the fluorescent indicator fluo-3 for assessment of intracellular free calcium ion (Ca[sup 2+]) concentrations with the aid of a confocal laser-scanning microscope. In the absence of added MgCI[sub 2], the addition of NMDA induced a rapid but sustained increase in the number of fluorescent neurons in a concentration-dependent manner at a concentration range of 1-100 µM with the increase by KCI being transient. The addition of FeCI[sub 2], but not FeCI[sub 3], markedly inhibited the increase by NMDA in a reversible manner at concentrations of 10-200 µM, without affecting that by KCI. Extensive analyses revealed clear differentiation between inhibitions by ferrous iron and other channel blockers known to date. The inhibition by FeCI[sub 2] was completely prevented by the addition of two different iron chelators. Exposure to NMDA alone did not lead to cell death in immature cultured neurons, however, while further addition of FeCI[sub 2] invariably induced neuronal cell death 24 h after exposure. These results give support to our previous proposal that NMDA receptor complex may contain a novel site sensitive to blockade by ferrous iron in rat brain. [ABSTRACT FROM AUTHOR]