Mitochondria show extensive movement along neuronal processes, but the mechanisms and function of this movement are not clearly understood. We have used high-resolution confocal microscopy to simultaneously monitor movement of mitochondria and changes in intracellular [Ca(2+)] ([Ca(2+)](i)) in rat cortical neurons. A significant percentage (27%) of the total mitochondria in cortical neuronal processes showed movement over distances of >2 microM. The average velocity was 0.52 microm/s. The velocity, direction, and pattern of mitochondrial movement were not affected by transient increases in [Ca(2+)](i) associated with spontaneous firing of action potentials. Stimulation of Ca(2+) transients with forskolin (10 microM) or bicuculline (10 microM), or sustained elevations of [Ca(2+)](i) evoked by glutamate (10 microM) also had no effect on mitochondrial transit. Neither removal of extracellular Ca(2+), depletion of intracellular Ca(2+) stores with thapsigargin, or inhibition of synaptic activity with TTX (1 microM) or a cocktail of CNQX (10 microM) and MK801 (10 microM) affected mitochondrial movement. These results indicate that movement of mitochondria along processes is a fundamental activity in neurons that occurs independently of physiological changes in [Ca(2+)](i) associated with action potential firing, synaptic activity, or release of Ca(2+) from intracellular stores.