We have used rapid confocal microscopy to investigate the mechanism of Ca(2+) signals in individual dendritic spines of hippocampal CA1 pyramidal cells. The experiments focused on the signals that occur during single weak synaptic responses that were subthreshold for triggering postsynaptic action potentials. These Ca(2+) signals were not strongly affected by blocking the EPSPs with the AMPA receptor antagonist CNQX. The signals were also not strongly reduced by blocking T-type voltage-gated Ca(2+) channels (VGCCs) with Ni(2+) or by blocking a broad range of VGCCs with intracellular D890. The spine Ca(2+) signals were blocked by NMDA receptor channel (NMDAR) antagonist and had the voltage dependence characteristic of these channels. Neither ryanodine nor cyclopiazonic acid (CPA), substances known to deplete intracellular Ca(2+) stores, substantially reduced the amplitude of synaptically evoked Ca(2+) signals. CPA slowed the recovery phase of Ca(2+) signals in spines produced by synaptic stimulation or by backpropagating action potentials, suggesting a role of intracellular stores in Ca(2+) reuptake. Thus, we find that Ca(2+) release from intracellular stores is not required to produce spine Ca(2+) signals. We conclude that synaptic Ca(2+) signals in spines are primarily caused by Ca(2+) entry through NMDARs. Although these channels are largely blocked by Mg(2+) at voltages near the resting potential, they can nevertheless produce significant Ca(2+) elevation. The resulting Ca(2+) signals are an integral component of individual evoked or spontaneous synaptic events and may be important in the maintenance of synaptic function.