An inhibitor of catalase accumulated when leaves of chilling-sensitive species were stored in the dark at 0 degrees C. The inhibitor could be removed from crude extracts by passing them through a column of Sephadex G-25. After this treatment, the catalase activity of extracts of chilled tissues was found to be equal to that of extracts from unchilled leaves. When chilled tissues were incubated at 20 degrees C, the inhibitor of catalase was lost, unless the tissues had been irreversibly damaged. It specifically inhibited plant catalase, and had no effect on mammalian catalase, plant malic dehydrogenase, or plant superoxide dismutase.Despite the presence of catalase inhibitor in extracts of chilled plants, no increase in the level of H(2)O(2) in chilled tissues was found, suggesting either that the inhibitor is compartmentalized and not in contact with catalase in vivo, or that the level of H(2)O(2) is controlled by means other than through catalase activity. Plant tissues normally contain H(2)O(2) which is destroyed by catalase when they are damaged. After chilling, H(2)O(2) leaking from already injured cells would not be so readily removed by the inhibited catalase, and could contribute to further injury by acting as a source of free radical oxidants.