Phosphoenolpyruvate carboxylases (PEPCs), one form of which in each legume species plays a central role in the carbon metabolism in symbiotic root nodules, are activated through phosphorylation of a conserved residue by a specific protein kinase (PEPC-PK). We characterized the cDNAs for two PEPC isoforms of Lotus japonicus, an amide-translocating legume that forms determinate nodules. One gene encodes a nodule-enhanced form, which is more closely related to the PEPCs in amide-type indeterminate nodules than those in ureide-type determinate nodules. The other gene is expressed in shoots and roots at a low level. Both forms have the putative phosphorylation site, Ser11. We also isolated a cDNA and the corresponding genomic DNA for PEPC-PK of L. japonicus. The recombinant PEPC-PK protein expressed in Escherichia coli phosphorylated recombinant maize C4-form PEPC efficiently in vitro. The level of mRNA for PEPC-PK was high in root nodules, and those in shoots and roots were also significant. In situ hybridization revealed that the expression patterns of the transcripts for PEPC and PEPC-PK were similar in mature root nodules, but were different in emerging nodules. When L. japonicus seedlings were subjected to prolonged darkness and subsequent illumination, the activity of PEPC-PK and the mRNA levels of both PEPC and PEPC-PK in nodules decreased and then recovered, suggesting that they are regulated according to the amounts of photosynthates transported from shoots.