Differences between antioxidant responses to drought in C-3 and C-4 plants are rather scanty. Even, we are not aware of any research on comparative ROS formation and antioxidant enzymes in C-3 and C-4 species differing in carboxylation pathway of same genus which would be useful to prevent other differences in plant metabolism. With this aim, relative shoot growth rate, relative water content and osmotic potential, hydrogen peroxide (H2O2) content and NADPH oxidase (NOX) activity, antioxidant defence system (superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR) enzymes and their isoenzymes), CAT1 mRNA level, and lipid peroxidation in seedlings of Cleome spinosa (C-3) and Cleome gynandra (C-4) species of Cleome genus exposed to drought stress for 5 and 10 day (d) were comparatively investigated. Constitutive levels of antioxidant enzymes (except SOD) were consistently higher in C. spinosa than in C. gynandra under control conditions. CAT1 gene expression in C. spinosa was correlated with CAT activity but CAT1 gene expression in C. gynandra at 10 d did not show this correlation. Drought stress caused an increase in POX, CAT, APX and GR in both species. However, SOD activity was slightly decreased in C. gynandra while it was remained unchanged or increased on 5 and 10 d of stress in C. spinosa, respectively. Parallel to results of malon dialdehyde (MDA), H2O2 content was also remarkably increased in C. spinosa as compared to C. gynandra under drought stress. These results suggest that in C. spinosa, antioxidant defence system was insufficient to suppress the increasing ROS production under stress condition. On the other hand, in C. gynandra, although its induction was lower as compared to C. spinosa, antioxidant system was able to cope with ROS formation under drought stress. (C) 2011 Elsevier Ireland Ltd. All rights reserved.