Abstract: Al tolerance is one of the main growth and yield limiting factor in world and mechanism for Al-tolerance or Al-sensitivity yet to be clarified. We previously reported Japonica rice (Oryza sariva L.) cultivar Rikuu-20 as Al-sensitive, whereas a closely related cultivar that is a descendant of Rikuu-20, Rikuu-132, was Al tolerant. The objective of the present study was to clarify the role of plasma membrane lipid layer for Al tolerance in rice. The previously stated two cultivars were compared to determine mechanisms underlying variations in Al tolerance. The sensitive cultivar Rikuu-20 showed increased permeability of the Plasma Membrane (PM) and greater Al uptake within 24 h of Al treatment. Lipid composition of the PM differed between these cultivars was considered to be the primary account for the difference in Al tolerance. The tolerant cultivar Rikuu-132 showed a less PM permeabilization and Al accumulation which was drastically decreased in presence of paclobutrazol, a sterol metabolism inhibitor which reduces Δ5-sterols and accumulates abnormal sterols by inhibiting obtusifoliol-14α-demethylase. The tolerant cultivar Rikuu-132 had lower phospholipids than that of sensitive cultivar Rikuu-20, suggesting that the PM of Rikuu-132 is less negatively charged and less permeabilized than that of Rikuu-20. We used inhibitor of Δ5-sterol synthesis to alter the ratio of phospholipids to Δ5-sterols in both cultivars. These inhibitors reduced Al tolerance in Rikuu-132 whereas Al tolerance of Rikuu-20 was unchanged suggesting that PM lipid composition greatly regulating Al tolerance in rice.