Several studies have shown that a prolonged Ca(2+) elevation follows a glutamate-mediated excitotoxic insult in cultured neurons, and may be associated with impending cell death. Recently, we showed that the prolonged Ca(2+) elevation that emerges as neurons age in culture is specifically linked to an age-related increase in excitotoxic vulnerability. However, the multiple sources of Ca(2+) that contribute to Ca(2+) elevation during and after glutamate exposure are not well understood. Here, we examined the Ca(2+) sources of the age-related prolonged Ca(2+) elevation in cultured hippocampal neurons. Studies with caffeine showed that the ryanodine receptor-dependent releasable pool of Ca(2+) from intracellular stores was similar in older and younger neurons. Thapsigargin, which inhibits intracellular store refilling, did not mimic the age-related prolonged Ca(2+) elevation and, in fact, partially reduced it. Ryanodine, which blocks Ca(2+)-induced Ca(2+)-release (CICR) from stores, completely blocked the age-related prolonged Ca(2+) elevation following glutamate exposure but did not alter maximal Ca(2+) elevation during the glutamate exposure. Thus, we conclude that sustained CICR plays a selective and key role in generating the lethal, age-related, prolonged Ca(2+) elevation, and is the likely mechanism underlying age-related, enhanced vulnerability to excitotoxicity in neurons.