We measured glutamate-stimulated increases in intracellular free Ca2+ concentrations ([Ca2+]i) in cultured rat forebrain neurons loaded with both a high- and a low-affinity Ca2+ indicator. In these dual-dye studies, the high-affinity indicators Fluo-3 and Fura-2 gave both qualitatively and quantitatively different results than the low-affinity indicators Mag-fura-2 and Calcium Green-5N. The glutamate-stimulated peak [Ca2+]i reported using Fluo-3 and Fura-2 were less than 6 microM while the low-affinity indicators Mag-fura-2 and Calcium Green-5N indicated [Ca2+]i responses were more than 12 microM. The shapes of the responses obtained with the two types of dyes were also different, and only the low-affinity indicators effectively demonstrated that [Ca2+]i continues to rise during prolonged (5-min) stimulations. These dual-dye studies also revealed that kainate- and depolarization-induced [Ca2+]i responses could be differentiated from glutamate responses only with the low-affinity indicators. These results suggest that Fluo-3 and Fura-2 underestimate [Ca2+]i induced by excitotoxic glutamate stimuli and that these responses are greater than have previously been reported. These studies also reveal, in contrast to previous reports, that excitotoxic stimuli do indeed cause increases in [Ca2+]i that are greater than those produced by non-toxic stimuli.