Although there has been a large body of literature from animal studies concerning neuronal excitatory amino acid (EAA) receptors and their possible roles in brain development, function, and pathology, essentially no direct information on actions of EAAs in humans has previously been available. We now report on experiments in cell cultured human embryonic cerebral cortical neurons which directly addressed the actions of EAAs in the developing human brain. In cultures established from 14-week fetuses, neurons were insensitive to glutamate neurotoxicity during the first 30 days in culture. After 30 days in culture increasingly more neurons became vulnerable to glutamate acting at the N-methyl-D-aspartate and kainate type receptors. The development of calcium responses to glutamate (as measured with the calcium indicator dye fura-2) preceded sensitivity to excitotoxicity by several weeks in the human neurons. Glutamate-induced rises in intracellular calcium and neurotoxicity developed much more rapidly in rat cortical neurons. Studies of dynamic aspects of calcium responses to calcium ionophore A23187 in human and rat cortical neurons demonstrated a direct relation between calcium buffering ability and resistance to EAA neurotoxicity. Interestingly, the human neurons were better able to buffer a calcium load than were rat neurons, suggesting that species-specific and/or developmental stage-specific differences in calcium-buffering systems are likely to play roles in determining neuronal vulnerability to EAAs. These initial observations indicate that human cortical neurons become sensitive to EAAs during the prenatal period, and suggest that EAAs may play important roles in both normal human brain development and neurodegenerative processes.