Carbonic anhydrase III (CA III; EC 4.2.1.1) is a cytoplasmic enzyme that exhibits a relatively low carbon dioxide hydratase activity. It is expressed at a very high level in skeletal muscle, where physical exercise has been shown to increase free radical production. In this work we show the effect of overexpression of CA III on cellular response to oxidative stress. Rat CA III cDNA was transfected to NIH/3T3 cells, which have no endogenous CA III expression. The isolated clones expressed CA III mRNA and protein. The protein was localized to cytoplasm and nuclei. Compared to parental cells, transfected cells showed lower basal oxidized state as judged by measurement of intracellular reactive oxygen species (ROS) using fluorescent dye and an image analysis system. Addition of exogenous H2O2 to cells induced a rapid increase of ROS in control but not in CA III overexpressing cells. Association of this phenomenon with CA III expression was further confirmed by showing that overexpression of CA II could not prevent H2O2-stimulated increase of ROS. In proliferation assays, CA III overexpressing cells grew faster and were more resistant to cytotoxic concentrations of H2O2 than control cells. After a 16 h exposure to oxidative stress, the number of apoptotic cells was also reduced in transfectants. Our results suggest that CA III functions as an oxyradical scavenger and thus protects cells from oxidative damage. A lower level of free radicals in CA III overexpressing cells may also affect growth signaling pathways.