Nitric oxide has been recognized in recent years as an important mediator of neuronal toxicity, which in many cases involves alterations of the cytoplasmic Ca2+ concentration ([Ca2+]i). In [Ca2+]i fluorimetric experiments on cultured hippocampal neurons, the nitric oxide-releasing agent S-nitrosocysteine produced a delayed rise in [Ca2+]i over a 20-min exposure, which was accompanied by a progressive slowing of the kinetics of recovery from depolarization-induced [Ca2+]i transients. These effects were blocked by oxyhemoglobin and by superoxide dismutase, confirming nitric oxide as the responsible agent, and suggesting that they involved peroxynitrite formation. Similar alterations of [Ca2+]i homeostasis were produced by the mitochondrial ATP synthase inhibitor oligomycin, and when an ATP-regenerating system was supplied via the patch pipette in combined whole-cell patch-clamp-[Ca2+]i fluorimetry experiments, S-nitrosocysteine had no effect on the resting [Ca2+]i or on the recovery kinetics of [Ca2+]i transients induced by direct depolarization. We conclude that prolonged exposure to nitric oxide disrupts [Ca2+]i homeostasis in hippocampal neurons by impairing Ca2+ removal from the cytoplasm, possibly as a result of ATP depletion. The resulting persistent alterations in [Ca2+]i may contribute to the delayed neurotoxicity of nitric oxide.