Iron is an essential element for cells but may also be an important cytotoxin. However, very little is known about iron transport, redox status, or toxicity specifically inside cells. In this study, we exploited the sensitivity of fura-2 to quenching by ferrous iron (Fe²⁺) to detect intracellular free iron ([Fe²⁺]_i) in neurons, astrocytes, and oligodendrocytes in primary culture. All cell types exposed to Fe²⁺in the presence of the ionophore pyrithione rapidly accumulated Fe²⁺to a similar extent. The heavy-metal chelators bipyridyl andN,N,N′,N′-tetrakis(2-pyridalmethyl)ethyl-enediamine rapidly reversed the increase in [Fe²⁺]_i, whereas desferrioxamine had little effect. Interestingly, the Fe²⁺-mediated quenching of fura-2 fluorescence was reversed in a concentration-dependent manner by hydrogen peroxide. This was likely caused by the oxidation of Fe²⁺to Fe³⁺inside the cell. Acute exposure of cells to Fe²⁺was only toxic when the metal was applied together with pyrithione, showing that Fe²⁺is only toxic when elevated inside cells. Interestingly, only neurons and oligodendrocytes were injured by this elevation in [Fe²⁺]_i, whereas astrocytes were unaffected, although [Fe²⁺]_iwas elevated to the same degree in each cell type. These studies provide a novel approach for detecting [Fe²⁺]_iin a manner sensitive to the redox state of the metal. These studies also provide a model system for the study of the toxic consequences of elevated [Fe²⁺]_iin neural cells.