Lysosomes are membrane bound structures that accumulate and hydrolyze material internalized by the endocytic pathway. A very conspicuous property of this subcellular compartment is its relatively high equilibrium density. The actual mechanism that regulates lysosomal density is poorly understood. In an attempt to gain knowledge on the factors that regulate lysosomal density we have assessed the equilibrium density of lysosomal markers after in vitro incubation of a lysosome-enriched subcellular fraction. Incubation at pH 6 for 10 min at 37 degrees C causes a density shift of several lysosomal markers to light density regions of Percoll gradients. Addition of ATP was able to prevent the acid-induced density shift. Pretreatment of the vesicles with N-ethylmaleimide (NEM) or trypsin inhibited the effect of ATP. Working in intact cells, ATP depletion, a condition that causes cytoplasmic acidification, also decreases lysosomal density. The results indicate that at low pH lysosomal density is preserved by an active process that requires ATP and membrane associated proteins.