Although rice (Oryza sativa) is the most Al-tolerant species among small-grain cereal crops, there is wide genotypic variation in its tolerance to Al toxicity. A number of quantitative trait loci (QTLs) for Al tolerance have been detected, but the responsible genes have not been identified. By using chromosome segment substitution lines, this work found that Nrat1, a gene encoding an Al transporter, is responsible for a QTL previously detected on chromosome 2. Substitution of the chromosome segment containing Nrat1 from Koshihikari (Al-tolerant variety) by that from Kasalath (Al-sensitive variety) decreased Nrat1 expression and Al uptake and tolerance, but increased binding of Al to the cell wall. Nrat1 in Kasalath showed tissue localization similar to Koshihikari in the roots. Although Koshihikari and Kasalath differed in four amino acids in Nrat1 protein, Nrat1 from Kasalath also showed transport activity for Al. Analysis with site-directed mutagenesis revealed that these differences did not affect the Al-transport activity much. Furthermore, there was no correlation between Al tolerance and the open-reading-frame sequence differences in other rice varieties. On the other hand, there was good correlation between Nrat1 expression and Al tolerance; however, sequence comparison of the promoter region up to 2.1 kb did not give a clear difference between the Al-tolerant and -sensitive varieties. Taken together, these results indicate that differential expression of Nrat1 is responsible for the QTL for Al tolerance on chromosome 2, although the mechanism controlling Nrat1 expression remains to be examined.