The effects of two aminoglycoside antibiotics, neomycin and Geneticin, on the endocytic pathway were studied using a cell-free assay that reconstitutes endosome-endosome fusion. Both drugs inhibit the rate and extent of endosome fusion in a dose-dependent manner with IC50 values of approximately 45 microM and approximately 1 mM, respectively. Because the IC50 for neomycin falls within the range of affinities reported for its binding to acidic phospholipids, notably phosphatidylinositol 4,5-bisphosphate (PIP2), these data suggest that negatively charged lipids are required for endosome fusion. A role for negatively charged lipids in membrane traffic has been postulated to involve the activity of a PIP2-dependent phospholipase D (PLD) stimulated by the GTP-binding protein ADP-ribosylation factor (ARF). Although neomycin blocks endosome fusion at a stage of the in vitro reaction that is temporally related to steps inhibited by cytosolic ARFs when they bind guanosine-5'-gamma-thiophosphate (GTPgammaS), these inhibitors appear to act in a synergistic manner. This idea is confirmed by the fact that addition of a PIP2-independent PLD does not suppress neomycin inhibition of endosome fusion; moreover, in vitro fusion activity is not affected by the pleckstrin homology domain of phosphoinositide-specific phospholipase C delta1, which binds to acidic phospholipids, particularly PIP2, with high affinity. Thus, although aminoglycoside-sensitive elements of endosome fusion are required at mechanistic stages that are also blocked by GTPgammaS-bound ARF, these effects are unrelated to inhibition of the PIP2-dependent PLD activity stimulated by this GTP-binding protein. These results argue that there are additional mechanistic roles for acidic phospholipids in the endosomal pathway.