We have shown that cholera toxin and cAMP greatly increase both acidification rates of liver endosomes and the liver and endosome content of fluid-phase endocytosis probes. In this study lysosomes were purified from control and cholera toxin-treated livers that were pulsed with fluorescein conjugated dextran and chased overnight. Cholera toxin-treated livers weighed less, contained less protein and exhibited higher contents of lysosomal marker enzymes, consistent with the catabolic effects of this agent. By contrast to its effects on endosomes, cholera toxin had no consistent or significant effect on lysosome acidification rates, steady-state internal pH or potassium content, proton leak rates or fluorescein-dextran content. We conclude that cholera toxin and cAMP predominantly alter earlier steps of endocytosis but may also increase transfer of probes from lysosomes to bile. Copyright 2000 Academic Press.