PIKfyve enzymatic activity is required in maintaining late endocytic membrane integrity. PIKfyve is a dual specificity enzyme that phosphorylates phosphatidylinositol (PtdIns) and PtdIns 3-P at the 5-hydroxyl and unidentified endogenous protein substrate(s). To determine which of these activities (lipid versus protein kinase activity) is responsible for endomembrane homeostasis we analyzed a double mutant PIKfyveK1999E/K2000E. These substitutions in the putative lipid-substrate activation loop nearly completely abrogated the lipid kinase activity without any significant effect on the protein kinase activity of PIKfyveK1999E/K2000E. Expression of PIKfyveK1999E/K2000E in COS cells induced a dramatic dominant-negative effect in the form of endomembrane swelling and vacuolation. In addition, the lipid-substrate specificity of PIKfyve was modified by introducing single mutations in Lys-1999 or Lys-2000. This yielded proteins with preferentially abrogated synthesis of PtdIns 5-P (PIKfyveK2000E) or PtdIns 3,5-P2 (PIKfyveK1999E), of which only the PIKfyveK1999E mutant induced the characteristic endomembrane defects upon cell transfection. Furthermore, phosphoinositide microinjection into cells demonstrated a selective ability of PtdIns 3,5-P2 to correct the endomembrane defects induced by the dominant-negative PIKfyve lipid kinase. deficient mutants. Thus, PtdIns 3,5-P2 production by PIKfyve is crucial for endomembrane integrity, and Lys-1999 most likely directs the PIKfyve interactions with the 3-phosphate group in PtdIns 3-P.