Highlight Photorespiratory genes are expressed tissue-specific in the leaves of the C sub(4) grass Sorghum bicolor. Most but not all of them are confined to the bundle sheath cells.One of the hallmarks of C sub(4) plants is the division of labor between two different photosynthetic cell types, the mesophyll and the bundle sheath cells. C sub(4) plants are of polyphyletic origin and, during the evolution of C sub(4) photosynthesis, the expression of thousands of genes was altered and many genes acquired a cell type-specific or preferential expression pattern. Several lines of evidence, including computational modeling and physiological and phylogenetic analyses, indicate that alterations in the expression of a key photorespiration-related gene, encoding the glycine decarboxylase P subunit, was an early and important step during C sub(4) evolution. Restricting the expression of this gene to the bundle sheath led to the establishment of a photorespiratory CO sub(2) pump. We were interested in whether the expression of genes related to photorespiration remains bundle sheath specific in a fully optimized C sub(4) species. Therefore we analyzed the expression of photorespiratory and C sub(4) cycle genes using RNA in situ hybridization and transcriptome analysis of isolated mesophyll and bundle sheath cells in the C sub(4) grass Sorghum bicolor. It turns out that the C sub(4) metabolism of Sorghum is based solely on the NADP-dependent malic enzyme pathway. The majority of photorespiratory gene expression, with some important exceptions, is restricted to the bundle sheath.