A peptide containing the N-terminal phosphorylation site (Ser-8) of Sorghum C4-phospho enolpyruvate carboxylase (PEPC) was synthesized, purified and used to raise an antiserum in rabbits. Affinity-purified IgGs prevented PEPC phosphorylation in a reconstituted in vitro assay and reacted with both the phosphorylated and dephosphorylated forms of either native or denatured PEPC in immunoblotting experiments. Saturation of dephospho-PEPC with these specific IgGs resulted in a marked alteration of its functional and regulatory properties that mimicked phosphorylation of Ser-8. A series of recombinant C4 PEPCs mutated in the N-terminal phosphorylation domain and a C3-like PEPC isozyme from Sorghum behaved similarly to their C4 counterpart with respect to these phosphorylation-site antibodies.