Myotubularins constitute a ubiquitous family of phosphatidylinositol (PI) 3-phosphatases implicated in several neuromuscular disorders. Myotubularin [myotubular myopathy 1 (MTM1)] PI 3-phosphatase is shown associated with early and late endosomes. Loss of endosomal phosphatidylinositol 3-phosphate [PI(3)P] upon overexpression of wild-type MTM1, but not a phosphatase-dead MTM1C375S mutant, resulted in altered early and late endosomal PI(3)P levels and rapid depletion of early endosome antigen-1. Membrane-bound MTM1 was directly complexed to the hVPS15/hVPS34 [vacuolar protein sorting (VPS)] PI 3-kinase complex with binding mediated by the WD40 domain of the hVPS15 (p150) adapter protein and independent of a GRAM-domain point mutation that blocks PI(3,5)P2 binding. The WD40 domain of hVPS15 also constitutes the binding site for Rab7 and, as shown previously, contributes to Rab5 binding. In vivo, the hVPS15/hVPS34 PI 3-kinase complex forms mutually exclusive complexes with the Rab GTPases (Rab5 or Rab7) or with MTM1, suggesting a competitive binding mechanism. Thus, the Rab GTPases together with MTM1 likely serve as molecular switches for controlling the sequential synthesis and degradation of endosomal PI(3)P. Normal levels of endosomal PI(3)P and PI(3,5)P2 are crucial for both endosomal morphology and function, suggesting that disruption of endosomal sorting and trafficking in skeletal muscle when MTM1 is mutated may be a key factor in precipitating X-linked MTM.