The C₄ photosynthetic pathway involves the assimilation of CO₂ by phosphoenolpyruvate carboxylase (PEPC) and the subsequent decarboxylation of C₄ acids. The enzymes of the CO₂ concentrating mechanism could be affected under water deficit and limit C ₄ photosynthesis. Three different C₄ grasses were submitted to gradually induced drought stress conditions: Paspalum dilatatum (NADP-malic enzyme, NADP-ME), Cynodon dactylon (NAD-malic enzyme, NAD-ME) and Zoysia japonica (PEP carboxykinase, PEPCK). Moderate leaf dehydration affected the activity and regulation of PEPC in a similar manner in the three grasses but had species-specific effects on the C₄ acid decarboxylases, NADP-ME, NAD-ME and PEPCK, although changes in the C₄ enzyme activities were small. In all three species, the PEPC phosphorylation state, judged by the inhibitory effect of l-malate on PEPC activity, increased with water deficit and could promote increased assimilation of CO₂ by the enzyme under stress conditions. Appreciable activity of PEPCK was observed in all three species suggesting that this enzyme may act as a supplementary decarboxylase to NADP-ME and NAD-ME in addition to its role in other metabolic pathways.