Cytosolic calcium ([Ca2+]i) is an important mediator of neuronal signal transduction, participating in diverse biochemical reactions that elicit changes in synaptic efficacy, metabolic rate, and gene transcription. Excessive [Ca2+]i also has been implicated as a cause of acute neuronal injury, although measurement of [Ca2+]i in living neurons by fluorescent calcium indicators has not consistently demonstrated a correlation between [Ca2+]i and the likelihood of neuronal death after a variety of potentially lethal insults. Using fluorescence videomicroscopy and microinjected calcium indicators, we measured [Ca2+]i in cultured cortical neurons during intense activation with either NMDA (300 microM) or AMPA (450 microM). At these concentrations NMDA killed >80% of the cultured neurons by the next day, whereas neuronal death from AMPA was <20%. Using the conventional calcium indicator, fura-2/AM, we estimated [Ca2+]i elevations to be approximately 300-400 nM during exposure to either glutamate agonist. In contrast, indicators with lower affinity for calcium, benzothiazole coumarin (BTC), and fura-2/dextran reported [Ca2+]i levels >5 microM during lethal NMDA exposure, but [Ca2+]i levels were <1.5 microM during nonlethal activation of AMPA receptors or voltage-gated calcium channels. Fura-2 reported [Ca2+]i responses during brief exposure to glutamate, NMDA, AMPA, kainate, and elevated extracellular K+ between 0.5 and 1 microM. With the use of BTC, only NMDA and glutamate exposures resulted in micromolar [Ca2+]i levels. Neurotoxic glutamate receptor activation is associated with sustained, micromolar [Ca2+]i elevation. The widely used calcium indicator fura-2 selectively underestimates [Ca2+]i, depending on the route of entry, even at levels that appear to be within its range of detection.