Neurodegeneration may occur secondary to glutamate-triggered Ca2+ influx through any of three routes: NMDA channels, voltage-sensitive Ca2+ channels (VSCC), and Ca(2+)-permeable AMPA/kainate channels (Ca-A/K). This study aims to examine Ca2+ ion dynamics in the generation of excitotoxic injury by correlating the relative amounts of 45Ca2+ that flow into cortical neurons through each of these routes over a 10 min epoch ("10 min Ca2+ loads;" a measure of influx rate), with resultant levels of intracellular free Ca2+ ([Ca2+]) and subsequent injury. Neurons possessing Ca-A/K make up a small subset (approximately 13%) of cortical neurons in culture, which can be identified by a histochemical stain based on kainate-stimulated Co2+ uptake (Co2+ (+) neurons) and which are unusually vulnerable to AMPA/kainate receptor-mediated injury. Initial studies using brief kainate exposures (to selectively destroy Co2+ (+) neurons) along with kainate-triggered 45Ca2+ influx measurements suggested that kainate causes rapid Ca2+ influx into Co2+ (+) neurons (comparable to that caused by NMDA). Influx through both Ca-A/K and NMDA channels increased proportionately with extracellular Ca2+, suggesting that these channels have high Ca2+ permeability. When cultures were subjected to exposures that gave similar 10 min Ca2+ loads through different routes, comparable levels of injury were observed, suggesting that net intracellular Ca2+ accumulation is a critical determinant of injury. However, the relationship between [Ca2+]i and influx was less direct: although exposures that gave the lowest or highest 10 min Ca2+ loads showed correspondingly lower or higher mean [Ca2+]i responses, there appears to be a wide range of exposures over which individual neuronal differences and sequestration/buffering mechanisms obscure [Ca2+]i as a reflection of influx rate.