Maintenance of an appropriate magnesium ion (Mg(2+)) concentration is essential for plant growth. In Arabidopsis thaliana, the CorA-MRS2-ALR-type proteins, named MRS2/MGT family proteins, are reportedly localized in various membranes and they function in Mg transport. However, knowledge of this family in other plant species is extremely limited. Furthermore, differential diversification among dicot and monocot plants suggested by phylogenetic analysis indicates that the role of the Arabidopsis MRS2/MGT family proteins is not the same in monocot plants. For a further understanding of this family in higher plants, functional analysis and gene expression profiling of rice MRS2/MGT family members were performed. A phylogenetic tree based on the isolated mRNA sequences of nine members of the OsMRS2 family confirmed that the MRS2/MGT family consists of five clades (A-E). A complementation assay in the yeast CM66 strain showed that four of the nine members possessed the Mg(2+) transport ability. Transient green fluorescent protein (GFP) expression in the isolated rice protoplast indicated that OsMRS2-5 and OsMRS2-6, belonging to clades D and A, respectively, localized in the chloroplast. Expression levels of these genes were low in the unexpanded yellow-green leaf, but increased considerably with leaf maturation. In addition, diurnal oscillation of expression was observed, particularly in OsMRS2-6 expression in the expanded leaf blade. We conclude that OsMRS2 family members function as Mg transporters and suggest that the genes belonging to clade A encode the chloroplast-localized Mg(2+) transporter in plants.