Real-time visualization of intracellular calcium concentration ([Ca2+]i) changes in mammalian Purkinje cells in vitro, utilizing the dye Fura-2, indicates that calcium action potentials are generated in the dendritic tree and follow a particular activation sequence. During spontaneous oscillations or after direct current injection, dendritic spikes are initiated as slow and graded plateau potentials at the level of the tertiary or spiny branchlets of the dendrite. As the plateau potentials become sufficiently high to reach the firing threshold for full dendritic spike generation, calcium entry is observed at the more proximal branches of the dendritic tree. These action potentials are then conducted orthodromically toward the soma and may invade other branches in the arbor antidromically. Simultaneous recording of the intracellular electrical activity and the Fura-2 fluorescent signal indicates that the intracellular calcium transients are accompanied by a very rapid increase in intracellular calcium concentration. This increase in [Ca2+]i exhibits an almost equally fast return to baseline after the termination of the action potential, indicating the presence of very efficient calcium sequestering and extruding mechanisms in the dendrites. Iontophoretic application of glutamate at the dendritic level provided a further demonstration of the spatial separation of plateau potentials from dendritic spikes and gives further insights into the details of dendritic integration in this neuron.