Subcellular fractionation of Balb/c 3T3 fibroblasts exposed to Wistaria floribunda agglutinin was performed to localize fractions containing internalized lectin. Employing two sequential self-generating silica sol density gradients, the postnuclear supernatant of cell homogenates was resolved into five distinct cellular components. Lysosome enzyme activities were displayed by two populations of vesicles, each separated from plasma membrane, golgi, and mitochondria markers. The more dense of these fractions exhibited morphological and biochemical properties ascribed to secondary lysosomes. The more buoyant population was similar to that reported by Rome et al [11] who noted that it may be a product of vesiculated golgi endoplasmic reticulum lysosome (GERL). Treatment with W floribunda agglutinin of cells, surface radioiodinated demonstrated that plasma membrane proteins were localized within both the buoyant and dense lysosome populations in as little as 10 min after exposure to lectin. Prolonged incubation of the cells with W floribunda agglutinin resulted in maintenance of this distribution. However, when nonradiactive cells were exposed to 125I-labeled W floribunda agglutinin for 10 min, radioactivity was detected only in the buoyant population of lysosomes as well as the plasma membrane/golgi fraction. Treatment of cells with W floribunda agglutinin for 30 min resulted in appearance of lectin associated radioactivity in the dense lysosome fraction in addition to those populations containing radioactivity seen after a 10-min incubation. These data indicate that the endocytosis of W floribunda agglutinin differed substantially from the internalization of a portion of the plasma membrane proteins. Furthermore, we found radioactivity associated with both plasma membrane proteins W floribunda agglutinin in regions of the density gradient fractionation that virtually lacked golgi and lysosome markers. These fractions may have represented populations of nonlysosome vesicles formed during the process of endocytosis.