Clathrin-coated vesicle endocytosis and macropinocytosis are distinct endocytic pathways demonstrable in several cell types including human epidermoid A431 cells (West, M.A., M.S. Bretscher, and C. Watts. 1989. J. Cell Biol. 109:2731-2739). Here we analyze the extent of mixing of macropinocytic endosome (macropinosome) content with that of conventional endosomes served by coated vesicle endocytosis. Using laser scanning confocal fluorescence microscopy we detected very little delivery of macropinosome content to either early or late endosomes-lysosomes as defined by labeling with transferrin or with LDL. Mixing of the contents of the macropinosomes and conventional endosomes was not induced by the addition of brefeldin A. Moreover, the morphology of macropinosomes was not grossly altered in the presence of brefeldin A, whilst in the same cells there were dramatic tubulation effects on conventional endosomes as reported by others. Although refractory to fusion with conventional endosomes, macropinosomes were nonetheless dynamic structures which sometimes exhibited vesiculo-tubular morphology in living cells and were capable of fusing with each other. We suggest that different endocytic mechanisms can give rise to distinct endosome populations.

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