Mitochondrial-inner-membrane vesicles, isolated from Ehrlich ascites carcinoma cells by titration with detergents, accumulated L-glutamine by a very efficient transport system. The vesicles lack any phosphate-activated glutaminase activity, allowing measurement of transport rates without interference by L-glutamine metabolism. The time course of the transport was linear for the first 60 s, reaching a steady state after 120 min. L-Glutamine transport showed co-operativity, with a Hill coefficient of 2.2; the kinetic parameters S0.5 and Vmax had values of 5 mM and 26 nmol/30 s per mg of protein respectively. The pH-dependence curve showed a bell shape, with a pH optimum about 8.0. The uptake of L-glutamine was not affected by the presence of a 50-fold molar excess of D-glutamine, L-cysteine, L-histidine, L-alanine, L-serine and L-leucine, whereas L-glutamate behaved as a poor inhibitor. The structural analogue L-glutamate gamma-hydroxamate (5mM) inhibited the net uptake by 68%; interestingly, other analogues (6-diazo-5-oxo-L-norleucine, acivicin and L-glutamate gamma-hydrazide) were ineffective. The impermeant thiol reagent p-chloromercuriphenylsulphonic acid (0.5mM) completely abolished the mitochondrial L-glutamine uptake; in contrast, other thiol reagents (mersalyl and N-ethylmaleimide) did not significantly affect the transport. These data confirm the existence of a specific transport system with high capacity for L-glutamine in the mitochondrial inner membrane, a step preceding the highly operative glutaminolysis in tumour cells.