Functionally distinct subpopulations of endosomes involved in targeting internalized material to specific intracellular destinations were resolved as two distinct peaks by free-flow electrophoresis. The less anodally shifted peak contained a population of early endosomes selectively labeled by brief exposures to endocytic tracers or by receptor-bound transferrin. Late endosomes, labeled only after longer periods of internalization, migrated more toward the anode. While both fluid phase and certain receptor-bound markers could be rapidly chased from early to late endosomes, transferrin remained in vesicles comigrating with early endosomes even after prolonged uptake. Thus early and late endosomes are kinetically related but functionally distinct: early endosomes serve as the major site of recycling of membrane and surface receptors and late endosomes are involved in delivery to lysosomes. The subpopulations each contain unique polypeptides not found on the plasma membrane. Thus, endosomes cannot be derived entirely from internalized cell surface components, and may have at least partly independent biosynthetic origins.