Manganese shares the uniport mechanism of mitochondrial calcium influx, accumulates in mitochondria and is cleared only very slowly from brain. Using dual-label isotope techniques, we have investigated both Mn2+ and Ca2+ mitochondrial efflux kinetics. We report that (1) there is no significant Na(+)-dependent Mn2+ efflux from brain mitochondria; (2) Mn2+ inhibits both Na(+)-dependent and Na(+)-independent Ca2+ efflux in brain, in a mode that appears to be primarily competitive and with apparent Ki values of 5.1 and 7.9 nmol/mg respectively; and (3) Ca2+ does not appear to inhibit Mn2+ efflux from brain mitochondria. Findings (1) and (2) suggest the possibility of mitochondrial accumulation of both Mn2+ and Ca2+ in Mn2(+)-intoxicated brain.