The light-dependent phosphorylation of the photosynthetic phosphoennlpyruvate carboxylase (PyrPC) was shown to occur in protoplasts from Sorghum mesophyll cells. I t was accompanied by an increase in PyrPC protein-serine-kinase activity and conferred the target-specific functional properties, i. e. an increase in V,,, and apparent Ki for L-malate, as previously found with the whole leaf. The light-dependent regulatory phosphorylation of PyrPC was (a) specifically promoted by the weak bases NH4C1 and methylamine (agents which increase cytosolic pH), but not by KN03, (b) inhibited by the cytosolic protein-synthesis inhibitor, cycloheximide, thus confirming that protein turnover is a component of the signal-transduction cascade, as reported in [4], (c) found to moderately decrease in the presence of EGTA and to be strongly depressed when the Ca2+-selective ionophore A23287 was added to the incubation medium together with EGTA. Addition of Ca2+, but not of Mg2 ', to the Ca2 '-depleted protoplasts partially, but significantly, relieved the inhibition. Calcium deprivation apparently affected the in-situ light-activation of the PyrPC protein kinase. These data indicated that both Ca2+ and an increase in cytosolic pH are required for the induction of PyrPC protein kinase activitylPyrPC phosphorylation in illuminated protoplasts from Sorghum mesophyll cells.In C4 plants, the primary photosynthetic C 0 2 fixation catalysed by phosphoenolpyruvate carboxylase (PyrPC) is a key step through which the control of carbon flow is operated. Regulation of the enzyme, in the cytosol of mesophyll cells, involves both antagonistic effectors i. e. glucose-6P, triose-P (positive), L-malate (negative), [I], and a light-dependent reversible phosphorylation process [2 -41. When measured at suboptimal conditions of pH and substrate PyrP (phosphoenolpyruvate) which are supposed to be physiological, the phosphorylated PyrPC displays an increased catalytic activity and Ki for L-malate. This post-translational modulation is thought to represent an additional mechanism allowing the Correspondence to J. Vidal, Laboratoire de Physiologic Vegetale MoICculaire, bbt. 430,