Pyruvate orthophosphate dikinase (PPDK, E.C. 2.7.9.1) is an abundant enzyme in the leaves of C4 plants associated with the dicarboxylic acid pathways of CO2 fixation in the dark. PPDK activity has also been detected in the seeds of maize and other, non-C4 cereals, where its role has yet to be established. Using an anti-PPDK serum, two cross-reacting species of M(r) close to 90 000 were detected in developing maize endosperm of wild-type plants. In two independent opaque-2 mutant lines, one of the polypeptides was absent and the other was reduced in level. Similarly, endosperm PPDK mRNA levels were greatly reduced in the opaque-2 maize lines compared to wild type, suggesting that endosperm PPDK gene expression is under Opaque-2 control. However, a low level of PPDK mRNA could still be detected in these mutants, indicating that PPDK gene expression is not absolutely dependent on Opaque-2 but rather can be modulated by it. This interpretation was reinforced by the demonstration that the distribution of PPDK transcripts is not affected in o2 mutants, although the level is reduced, and that PPDK mRNA is detectable prior to 02 mRNA during the maturation of wild-type maize endosperm. Using oligonucleotides specific for the different maize PPDK genes, the o2 mutations were shown to affect only cyPPDKZml gene expression in maize line A69Y.