1. Changes in cytosolic and mitochondrial [Ca2+] produced by brief trains of action potentials were measured in motor nerve terminals using a rapidly scanning confocal microscope. Cytosolic [Ca2+] was measured using ionophoretically injected Oregon Green BAPTA 5N (OG-5N). Mitochondrial [Ca2+] was measured using rhod-2, bath loaded as dihydrorhod-2. 2. In response to 100-250 stimuli at 25-100 Hz the average cytosolic [Ca2+] showed an initial rapid increase followed by a much slower rate of increase. Mitochondrial [Ca2+] showed no detectable increase during the first fifteen to twenty stimuli, but after this initial delay also showed an initially rapid rise followed by a slower rate of increase. The onset of the increase in mitochondrial [Ca2+] coincided with the slowing of the rate of rise of cytosolic [Ca2+]. The peak levels of cytosolic and mitochondrial [Ca2+] both increased with increasing frequencies of stimulation. 3. When stimulation terminated, the initial rate of decay of cytosolic [Ca2+] was much more rapid than that of mitochondrial [Ca2+]. 4. After addition of carbonyl cyanide m-chlorophenyl hydrazone (CCCP, 1-2 microM) to dissipate the proton electrochemical gradient across the mitochondrial membrane, cytosolic [Ca2+] rose rapidly throughout the stimulus train, reaching levels much higher than normal. CCCP inhibited the increase in mitochondrial [Ca2+]. 5. These results suggest that mitochondrial uptake of Ca2+ contributes importantly to buffering presynaptic cytosolic [Ca2+] during normal neuromuscular transmission.