We investigated the possible involvement of Al(3+)-induced alterations in membrane physical properties in Al(3+)-mediated inhibition of polyphosphoinositide (PPI) hydrolysis by the enzyme phosphatidylinositol-specific phospholipase C (PI-PLC). Liposomes composed of brain phosphatidylcholine (PC) or of PC and a mixture of brain PPI (PC:PPI) were incubated in the presence of Al(3+) (1-100 microM). We evaluated: (1) the amount of membrane-bound Al(3+), (2) the effects of Al(3+) on key membrane physical properties (surface potential, lipid fluidity, and lipid arrangement), and (3) the hydrolysis of PPI. Al(3+) binding to PC:PPI (60:40 mol/mol) liposomes was 1.3 times higher than to PC:PPI (90:10 mol/mol) liposomes and did not change after treatment with Triton X-100. Al(3+) increased membrane surface potential, promoted the loss of membrane fluidity, and caused lateral phase separation in PC:PPI liposomes. Phosphatidylinositol and phosphatidylinositol monophosphate hydrolysis in the presence of PI-PLC was not affected by Al(3+), but a significant and concentration-dependent inhibition of PIP(2) hydrolysis was observed, an effect that was prevented by previous bilayer disruption with Triton X-100. The obtained results support the hypothesis that Al(3+) binding to liposomes promotes the formation of rigid clusters enriched in PPI, restricting the accessibility of the enzyme to the substrate and subsequently inhibiting PIP(2) hydrolysis by PI-PLC.