We have isolated full-size cDNA sequences encoding the photosynthetic isoform of pyruvate orthophosphate dikinase (PPDK) of the C3 plant Flaveria pringlei. The encoded protein shares 96% identical amino acid residues with the C4 isoform of PPDK in the C4 species F. trinervia. The differing amino acid residues are evenly distributed along the polypeptide chain. Genomic Southern analysis of photosynthetic PPDK sequences in F. pringlei (C3), F. chloraefolia (C3-C4), F. linearis (C3-C4), F. floridana (C3-C4), F. brownii (C4-like) and F. trinervia (C4) reveals a simple hybridization pattern which is suggestive of a single gene. Northern hybridization experiments show that the abundance of PPDK transcripts in leaves correlates with the degree of C4 characteristics expressed in the various photosynthetic types analysed. This finding demonstrates that the increase in expression levels must have played a crucial role in evolving the C4-PPDK gene in the genus Flaveria.