The submersed monocot Hydrilla verticillata (L.f.) Royle is a facultative C(4) plant. It typically exhibits C(3) photosynthetic characteristics, but exposure to low [CO(2)] induces a C(4) system in which the C(4) and Calvin cycles co-exist in the same cell and the initial fixation in the light is catalyzed by phosphoenolpyruvate carboxylase (PEPC). Three full-length cDNAs encoding PEPC were isolated from H. verticillata, two from leaves and one from root. The sequences were 95% to 99% identical and shared a 75% to 85% similarity with other plant PEPCs. Transcript studies revealed that one isoform, Hvpepc4, was exclusively expressed in leaves during C(4) induction. This and enzyme kinetic data were consistent with it being the C(4) photosynthesis isoform. However, the C(4) signature serine of terrestrial plant C(4) isoforms was absent in this and the other H. verticillata sequences. Instead, alanine, typical of C(3) sequences, was present. Western analyses of C(3) and C(4) leaf extracts after anion-exchange chromatography showed similar dominant PEPC-specific bands at 110 kD. In phylogenetic analyses, the sequences grouped with C(3), non-graminaceous C(4), and Crassulacean acid metabolism PEPCs but not with the graminaceous C(4), and formed a clade with a gymnosperm, which is consistent with H. verticillata PEPC predating that of other C(4) angiosperms.