With an aim to clone the sorghum fertility restorer gene Rf1, a high-resolution genetic and physical map of the locus was constructed. The Rf1 locus was resolved to a 32-kb region spanning four open reading frames: a plasma membrane Ca(2+)-ATPase, a cyclin D-1, an unknown protein, and a pentatricopeptide repeat (PPR13) gene family member. An approximately 19-kb region spanning the cyclin D-1 and unknown protein genes was completely conserved between sterile and fertile plants as was the sequence spanning the coding region of the Ca(2+)-ATPase. In contrast, 19 sequence polymorphisms were located in an approximately 7-kb region spanning PPR13, and all markers cosegregated with the fertility restoration phenotype. PPR13 was predicted to encode a mitochondrial-targeted protein containing a single exon with 14 PPR repeats, and the protein is classified as an E-type PPR subfamily member. To permit sequence-based comparison of the sorghum and rice genomes in the Rf1 region, 0.53 Mb of sorghum chromosome 8 was sequenced and compared to the colinear region of rice chromosome 12. Genome comparison revealed a mosaic pattern of colinearity with an approximately 275-kb gene-poor region with little gene conservation and an adjacent, approximately 245-kb gene-rice region that is more highly conserved between rice and sorghum. Despite being located in a region of high gene conservation, sorghum PPR13 was not located in a colinear position on rice chromosome 12. The present results suggest that sorghum PPR13 represents a potential candidate for the sorghum Rf1 gene, and its presence in the sorghum genome indicates a single gene transposition event subsequent to the divergence of rice and sorghum ancestors.