Glycine decarboxylase (GDC) plays an important role in the photorespiratory metabolism of plants. GDC is composed of four subunits (P, H, L, and T) with the P-subunit (GLDP) serving as the actual decarboxylating unit. In C(3) plants, GDC can be found in all photosynthetic cells, whereas in leaves of C(3)-C(4) intermediate and C(4) species its occurrence is restricted to bundle-sheath cells. The specific expression of GLDP in bundle-sheath cells might have constituted a biochemical starting point for the evolution of C(4) photosynthesis. To understand the molecular mechanisms responsible for restricting GLDP expression to bundle-sheath cells, we performed a functional analysis of the GLDPA promoter from the C(4) species Flaveria trinervia. Expression of a promoter-reporter gene fusion in transgenic plants of the transformable C(4) species Flaveria bidentis (C(4)) showed that 1,571 bp of the GLDPA 5' flanking region contain all the necessary information for the specific expression in bundle-sheath cells and vascular bundles. Interestingly, we found that the GLDPA promoter of F. trinervia exhibits a C(4)-like spatial activity also in the C(3) plant Arabidopsis (Arabidopsis thaliana), indicating that a mechanism for bundle-sheath-specific expression is also present in this C(3) species. Using transgenic Arabidopsis, promoter deletion studies identified two regions in the GLDPA promoter, one conferring repression of gene expression in mesophyll cells and one functioning as a general transcriptional enhancer. Subsequent analyses in transgenic F. bidentis confirmed that these two segments fulfill the same function also in the C(4) context.