Calcium is an important second messenger in eukaryotic cells that regulates many different cellular processes. To elucidate calcium regulation in chloroplasts, we identified the targets of calcium-dependent phosphorylation within the stromal proteome. A 73 kDa protein was identified as one of the most dominant proteins undergoing phosphorylation in a calcium-dependent manner in the stromal extracts of both Arabidopsis and Pisum. It was identified as TKL (transketolase), an essential enzyme of both the Calvin-Benson-Bassham cycle and the oxidative pentose phosphate pathway. Calcium-dependent phosphorylation of both Arabidopsis isoforms (AtTKL1 and AtTKL2) could be confirmed in vitro using recombinant proteins. The phosphorylation is catalysed by a stroma-localized protein kinase, which cannot utilize GTP. Phosphorylation of AtTKL1, the dominant isoform in most tissues, occurs at a serine residue that is conserved in TKLs of vascular plants. By contrast, an aspartate residue is present in this position in cyanobacteria, algae and mosses. Characterization of a phosphomimetic mutant (S428D) indicated that Ser428 phosphorylation exerts significant effects on the enzyme's substrate saturation kinetics at specific physiological pH values. The results of the present study point to a role for TKL phosphorylation in the regulation of carbon allocation.