Glycerate 3-kinase (GLYK) is the terminal enzyme of the photorespiratory cycle in plants and many cyanobacteria. For several C(4) plants, notably grasses of the NADP-malic enzyme (ME) subtype, redox regulation of GLYK has been reported, but the responsible molecular mechanism is not known. We have analyzed the enzyme from the NADP-ME C(4) plant maize (Zea mays) and found that maize GLYK, in contrast to the enzyme from C(3) plants and a dicotyledonous NADP-ME C(4) plant, harbors a short carboxy-terminal extension. In its oxidized (night) form, a disulfide bridge is formed between the two cysteine residues present in this extra domain, and GLYK activity becomes inhibited. Cleavage of this bond by thioredoxin f produces the fully active thiol form, releasing autoinhibition. Fusion of the maize GLYK redox-regulatory domain to GLYK from C(3) plants confers redox regulation to these otherwise unregulated enzymes. It appears that redox regulation of GLYK could be an exclusive feature of monocotyledonous C(4) plants of the NADP-ME type, in which linear electron transport occurs only in the mesophyll chloroplasts. Hence, we suggest that GLYK, in addition to its function in photorespiration, provides glycerate 3-phosphate for the accelerated production of triose phosphate and its export from the mesophyll. This could facilitate the activation of redox-regulated Calvin cycle enzymes and the buildup of Calvin cycle intermediates in the bundle sheath of these particular C(4) plants during the dark/light transition.