The presence of calcium stores and their function in dendritic spines is still unsettled. We have now studied the kinetics of calcium released inside dendritic spines of cultured rat hippocampal neurons by flash photolysis of caged calcium. Photolysis of calcium produced a fast rise in [Ca2+](i), followed by a variable decay. We were able to correlate the decay of elevated [Ca2+](i) with the presence of synaptopodin (SP), an actin-binding protein, in the spines; spines containing SP generated the same initial [Ca2+](i) transient, but their decay time was significantly slower and more complex than that of SP-negative ones. The altered decay kinetics of the flash-elevated [Ca2+](i) transient was blocked by thapsigargin or cyclopiazonic acid (CPA), indicating that this kinetic change is due to compartmentalized release of calcium from intracellular stores. Thus, SP plays a pivotal role in the calcium store-associated ability of spines to locally tune calcium kinetics.