It is commonly admitted that the intralysosomal hydrolysis of thyroglobulin, which promotes thyroid hormonal secretion, requires an acidic pH. As a consequence the mechanisms by which such an acidic pH is set-up and maintained present an acute physiological interest. In the present work the extent of the intralysosomal hydrolysis of thyroglobulin (amount of TCA-soluble radioiodine formed during incubations of isolated thyroid lysosomes) has been retained as an index for the intralysosomal pH. Incubations in highly concentrated (250 mM) alkaline buffer, preincubations at 0 degree C in the presence of alkaline buffers or of methylamine, and incubations in the presence of FCCP (protonophore which dissipates proton-gradients) induced alkalinization of the lysosomal matrix (significant inhibition of thyroglobulin degradation). Addition of ATP to the incubation medium was shown to prevent these alkalinizations and to re-establish the acidic intralysosomal pH (normal thyroglobulin degradation) every time it had been experimentally increased. These results argue in favour of the existence of an ATP-driven proton pump in the membranes of thyroid lysosomes.