Mitochondria isolated from the ascites form of L1210 mouse leukemia cells readily accumulate Ca(2+) from the suspending medium and eject H(+) during oxidation of succinate in the presence of phosphate and Mg(2+), with normal stoichiometry between Ca(2+) uptake and electron transport. Ca(2+) loads up to 1600 ng-atoms per mg of protein are attained. As is the case in mitochondria from normal tissues, Ca(2+) uptake takes precedence over oxidative phosphorylation. However, Ca(2+) transport by the L-1210 mitochondria is unusual in other respects, which may possibly have general significance in tumor cells. The apparent affinity of the L1210 mitochondria for Ca(2+) in stimulation of oxygen uptake is about 3-fold greater than in normal liver mitochondria; moreover, the maximal rate of Ca(2+) transport is also considerably higher. Furthermore, when Ca(2+) pulses are added to L1210 mitochondria in the absence of phosphate or other permeant anions, much larger amounts of Ca(2+) are bound and H(+) ejected per atom of oxygen consumed than in the presence of phosphate; up to 7 Ca(2+) ions are bound per pair of electrons passing each energy-conserving site of the electron-transport chain. Such "superstoichiometry" of Ca(2+) uptake can be accounted for by two distinct types of respiration-dependent interaction of Ca(2+) with the L1210 mitochondria. One is the stimulation of oxygen consumption, which is achieved by relatively low concentrations of Ca(2+) (K(m) congruent with 8 muM) and is accompanied by binding of Ca(2+) up to 40 ng-atoms per mg of protein. The second process, also dependent on electron transport, is the binding of further Ca(2+) from the medium in exchange with previously stored membrane-bound protons, in which the affinity for Ca(2+) is much lower (K(m) congruent with 120 muM).