Here we describe a fast and efficient subcellular fractionation procedure that permits lysosomes to be separated from endosomes. Differential centrifugation is used to isolate a subcellular fraction containing both endosomes and lysosomes. Because lysosomes are sensitive to osmotic stress, hypotonic conditions destroy them, whereas endosomes, which are osmotically insensitive, stay intact. We demonstrate that hypotonic lysis of an endosome-lysosome-pool releases 85% of the lysosomes into the supernatant as measured by the activity of the lysosomal marker enzyme N-acetyl-beta-D-glucosaminidase (beta-AGA). The endosomal fraction is thoroughly characterised using a variety of subcellular markers. After pulsing cells with fluorescein isothiocyanate labelled transferrin (FITC-Tf), only about 12% of the marker is released under hypotonic conditions. A typical fractionation procedure takes about 1-2 h from initial cell homogenisation. The fractionation gives a pure lysosomal fraction (fraction L) containing high activities of lysosomal enzymes and an endosomal fraction (fraction E) reflecting different stages of endosomes.