Ca²⁺ channels and pyramidal cell Ca²⁺ are involved in hippocampal spreading depression (SD), but their roles remain elusive. Accordingly, we characterized Ca²⁺ changes during SD in CA3 pyramidal neurons and determined whether Ca²⁺ channel antagonists could prevent SD. SD was induced in hippocampal organotypic cultures (HOTCs), in which experimental conditions can be rigorously controlled. SD was triggered by transient exposure to sodium acetate (NaAc)‐based Ringer's coupled to an electrical pulse in the dentate gyrus and its occurrence confirmed with interstitial DC recordings. Pyramidal cell Ca²⁺ was measured with fura‐2 filled cells and was quantified at the soma, proximal and more distal apical dendrites. Regional Ca²⁺ changes began simultaneously with the triggering pulse of SD and reached three distinct peaks before returning to baseline concomitant with the interstitial DC potential of SD. The first peak occurred within 5 s of the triggering pulse, was smallest, and heralded the onset of SD. The second Ca²⁺ change was the greatest and reached a peak 6 s later, during the early phase of SD. The third was intermediate in size and occurred 18 s later, as SD reached its maximum interstitial DC change. SD was prevented by nonselective Ca²⁺ blockade (Ni²⁺ and Cd²⁺) but not by either L‐Ca²⁺ channel (nifedipine) or N‐Ca²⁺ channel inhibition (ω‐conotoxin GVIA). Importantly, SD was blocked by P/Q Ca²⁺ channel antagonism (ω‐agatoxin‐IVA), which also prompted a significant reduction in pyramidal cell Ca²⁺ change and hyperexcitability. These results show that the spatiotemporal pattern of pyramidal cell Ca²⁺ change with SD is multiphasic; they provide further evidence that these changes begin before electrophysiologic evidence of SD. Furthermore, they show that P/Q Ca²⁺ channel antagonism can prevent SD in HOTCs and it appears to do so by preventing the NaAc‐induced increased pyramidal cell excitability from NaAc exposure, which may involve altered GABAergic transmission. © 2003 Wiley‐Liss, Inc.