Hippocalcin is a Ca(2+)-binding protein, which belongs to the family of neuronal Ca(2+) sensors. It is highly expressed in the hippocampus but molecular mechanisms underlying its action in this part of the brain have not been investigated in detail. To study whether intrinsic neuronal activity could result in hippocalcin-mediated signal transduction we examined spontaneous and action potential (AP)-dependent changes in fluorescence of yellow fluorescent protein-tagged hippocalcin (HPCA-YFP) in transiently transfected hippocampal cultured neurons. In 6-12 DIV neurons HPCA-YFP spontaneously translocated longitudinally to specific sites within diffusionally confined domains of neuronal processes. The translocations to these sites were expressed as fast, reversible increases in HPCA-YFP fluorescence coincided with a decrease in adjacent sites indicating genuine protein translocation. Physiologically relevant neuronal stimulation with short trains of action potentials also resulted in fast, simultaneous, reversible, and [Ca(2+)](i)-dependent translocations of HPCA-YFP to several sites synchronizing hippocalcin signaling in different parts of neuronal processes. The amount of translocated protein increased with the number of action potentials in a train decoding the number of APs into the amount of translocated protein. We conclude that hippocalcin may signal within diffusionally restricted domains of neuronal processes in which it might play a physiological role in Ca(2+)-dependent local activation of specific molecular targets.