We established an in vitro cell-free system with which to evaluate the effects of basic substances and acidic ionophores on the internal pH and integrity of FITC-dextran (FD)-loaded lysosomes isolated from the rat liver. In this system, basic substances and acidic ionophores not only increased the internal pH dose-dependently, but also disrupted the lysosomes in the presence of Mg-ATP, which was detected as the release of FD from lysosomes. All of the vacuoligenic bases and acidic ionophores, but none of the non-vacuoligenic bases or neutral ionophores disrupted the lysosomes, suggesting that this phenomenon is an vitro manifestation of vacuole formation induced in vivo by basic substances and acidic ionophores. Lysosome disruption required a functional proton pump as well as permeant anions. It was inhibited by inhibitors of the lysosomal proton pump, including bafilomycin A1, N-ethylmaleimide (NEM), and N, N'-dicyclohexylcarbodiimide (DCCD), or when permeant anions were replaced with impermeant anions. It was also suppressed by increasing the osmotic pressure of the surrounding medium, suggesting that it was caused by osmotic swelling of lysosomes induced by protonated bases or cations characteristic of particular ionophores that accumulated within lysosomes driven by the proton pump. Furthermore, this lysosomal disruption was inhibited by cytosolic factors. This phenomenon will provide an in vitro system for studies on osmoregulation and the intracellular dynamics of the lysosomal system, including membrane fusion.