Fluorescein isothiocyanate-conjugated dextran (FITC-dextran) is internalized by endocytosis into the lysosome-like vacuoles of Saccharomyces cerevisiae (Makarow, M., 1985, EMBO (Eur. Mol. Biol. Organ.) J. 4:1861-1866). Here we show that under energy depletion conditions FITC-dextran accumulated in a cytoplasmic compartment, from which it could be chased to the vacuole when the energy block was removed. The internal pH of the intermediate compartment under energy depletion was determined by fluorometry to be 5.8. The pH could be raised by the lysosomotropic agent ammonium chloride, the protonophore carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (CCCP) and the ATPase inhibitors dicyclohexylcarbodiimide (DCCD) and sodium vanadate. The pH of the vacuole was found to be 6.5. It was raised by ammonium chloride, CCCP, and DCCD, but not with sodium vanadate. Efrapeptin had no effect on the internal pH of either compartment. By dissecting the endocytic pathway, two portions of the route leading to the vacuole could be studied separately. The internalization of FITC-dextran from the extracellular fluid to the intermediate compartment followed linear kinetics, was independent of energy, and occurred at temperatures of between 15 degrees and 37 degrees C. Transfer of the marker from the intermediate compartment to the vacuole required energy, took place at temperatures between 19 degrees and 37 degrees C, and had a half-time of 7 min at 37 degrees C. Transport of the marker from the exterior of the cell to the vacuole did not require acidic pH values in the intermediate compartment or the vacuole. We suggest that the cytoplasmic compartment revealed by FITC-dextran, under energy depletion, represents the equivalent of the endosomes of mammalian cells.