Phosphorylation of ADP and hydrolysis of ATP by isolated mitochondria from Ehrlich ascites tumuor cells is greatly reduced when the mitochondria have been preloaded with Ca 2+ (50 nmol/mg protein or more). Translocation of ADP is diminished in Ca 2+-loaded mitochondria. However, ATPase in toluene-permeabilized mitochondria and in inside-out submitochondrial particles is also strongly inhibited by micromolar concentrations of Ca 2+, indicating that, independently of adenine nucleotide transport, F 1F 0-ATPase is also affected. ATP hydrolysis by submitochondrial particles depleted of the inhibitory subunit of F 1F 0-ATPase is also inhibitor) is insensitive to Ca 2+; however, this sensitivity is restored when the particles are supplemented with the inhibitory subunit isolated from beef heart mitochondria. In view of the previous observations that glucose elicits in Ehrlich ascites tumour cells an increase of cytoplasmic free Ca 2+ (Teplova, V.V., Bogucka, K., Czy/.z, A., Evtodienko, YuV., Duszyński, J. and Wojtczak, L. (1003) Biochem. Biophys. Res. Commun. 196, 1148–1154) and that this calcium is then taken up by mitochondria, resulting in a strong inhibition of coupled respiration (Evtodienko, Yu.V.,Teplova, V.V., Duszyński, J., Bogucka, K. and Wojtczak, L. (1994) Cell Calcium 15, 439–446), the present results are discussed in terms of the mechanism of the Crabtree effect in tumour cells.