Previously (Singer, B., and Riezman, H. (1990) J. Cell Biol. 110, 1911-1922), we provided evidence for the existence of an endocytic intermediate(s) from the yeast Saccharomyces cerevisiae that is responsible for the transport of the pheromone alpha-factor from the plasma membrane to the vacuole. Here we show by kinetic analysis that the endocytic apparatus of yeast is composed of early and late endosomes, similar to what has been found in animal cells. We have developed a three-step isolation procedure to purify early and late endosomes, consisting of differential centrifugation, flotation on a Nycodenz density gradient, and sedimentation density gradient centrifugation on sucrose/D2O. Using internalized 35S-alpha-factor as a marker, the endosomal fractions were substantially enriched over other membranes, except for Golgi elements and a compartment containing binding protein. These contaminants could not be removed by other standard purification methods. We have analyzed the protein composition of our most pure early and late endosome fractions. By two-dimensional gel analysis we identified more than 20 proteins spots that are highly enriched in the early/late endosomal fractions. N-terminal protein sequencing resulted in the identification of four novel proteins.