Ascorbate peroxidase (AP) is a key enzyme that scavenges potentially harmful H2O2 and thus prevents oxidative damage in plants, especially in N2-fixing legume root nodules. The present study demonstrates that the nodule endodermis of alfalfa (Medicago sativa) root nodules contains elevated levels of AP protein, as well as the corresponding mRNA transcript and substrate (ascorbate). Enhanced AP protein levels were also found in cells immediately peripheral to the infected region of soybean (Glycine max), pea (Pisum sativum), clover (Trifolium pratense), and common bean (Phaseolus vulgaris) nodules. Regeneration of ascorbate was achieved by (homo)glutathione and associated enzymes of the ascorbate-glutathione pathway, which were present at high levels. The presence of high levels of antioxidants suggests that respiratory consumption of O2 in the endodermis or nodule parenchyma may be an essential component of the O2-diffusion barrier that regulates the entry of O2 into the central region of nodules and ensures optimal functioning of nitrogenase.Work done at Reed College and West Virginia State College was supported by National Science Foundation grant no. IBN-9507491. Additionally, partial support was provided by a grant from the Howard Hughes Medical Institute to the Reed College Biology Department under the 1991 Undergraduate Biological Sciences Initiative. Work done at Consejo Superior de Investigaciones Científicas (Zaragoza) was supported by grant no. PB95-0091 from Dirección General de Enseñanza Superior (Ministry of Education and Culture, Spain).Peer reviewed