We isolated two cDNAs of phosphoenolpyruvate carboxylase (PEPC) from developing rice seeds, Osppc1 and Osppc3. The deduced amino acid sequences of both cDNAs share several conserved motifs with other non-photosynthetic PEPCases, and these common motifs are known to be functionally important to their regulatory properties. The deduced protein sequence of Osppc1 was clustered into a monocotyledonous plant-specific clade, and Osppc3 was clustered into a gramineous plant-specific clade in the phylogenetic tree of plant PEPCases. The mRNA accumulations of Osppc1 and Osppc3 were found in developing rice seeds throughout the grain-filling stages, although their expression patterns differed: Osppc1 was strongly expressed at 7 d after flowering, and Osppc3 was strongly expressed at 4 d after flowering. The kinetic properties of the Osppc1 recombinant protein were quite similar to those of maize root-type PEPCase, except that the sensitivity for malate at pH 7.3 was weaker. Mining rice microarray data, we observed that Osppc1 was co-expressed with aspartate aminotransferase and alanine aminotransferase, which are involved in seed nitrogen metabolism. Moreover, reannotation of the co-expressed genes revealed that Osppc1, the two aminotransferases and the enolase were mapped on to the consecutive reaction from 2-phosphoglycerate to glutamate and pyruvate in the cytosol. These results imply that Osppc1 functions cooperatively with the two aminotransferases in the synthesis of amino acids that are used for storage protein synthesis in developing rice seeds.