The mammalian phosphatidylinositol (PtdIns) 5-P/PtdIns 3,5-P2-producing kinase PIKfyve has been implicated in maintaining endomembrane homeostasis in mammalian cells. To address the role of PIKfyve in trafficking processes, we examined the functioning of the biosynthetic, endocytic, and recycling pathways in stable human embryonic kidney 293 cell lines inducibly expressing the wild-type or kinase-defective dominant-negative form. PIKfyveWT or PIKfyveK1831E expression did not affect the processing and lysosomal targeting of newly synthesized procathepsin D. Likewise the rates of transferrin uptake/recycling or epidermal growth factor receptor degradation were not altered upon expression of either protein. In contrast, PIKfyveK1831E but not PIKfyveWT expression markedly impaired the late uptake of fluid phase marker horseradish peroxidase. Inspection of the organelle morphology by confocal microscopy with specific markers in COS cells transiently expressing PIKfyveK1831E showed the Golgi apparatus, end lysosomes, and the recycling compartment indistinguishable from nontransfected cells, despite the dramatic PIKfyveK1831E-induced endomembrane vacuolation. In contrast, we observed a striking effect on the late endocytic compartment, marked by disruption of the dextran-labeled perinuclear endosomal compartment and formation of dispersed enlarged vesicles. Electron microscopy identified the cytoplasmic vacuoles in the PIKfyveK1831E-expressing human embryonic kidney 293 cells as enlarged multivesicular body-like structures with substantially lower number of internal vesicles and membrane whorls. Together, these data indicate that PIKfyve selectively regulates the sorting and traffic of peripheral endosomes containing lysosomaly directed fluid phase cargo through controlling the morphogenesis and function of multivesicular bodies.