Ascorbate is the most abundant small molecule antioxidant in plants and is proposed to function, along with other members of an antioxidant network, in controlling reactive oxygen species. A biochemical and molecular characterization of four ascorbate-deficient (vtc) Arabidopsis thaliana mutants has been carried out to determine if ascorbate deficiency is compensated by changes in the other major antioxidants. Seedlings grown in vitro were used to minimize stress and longer term developmental differences. Comparison was made with the low glutathione cad2 mutant and vtc2-1 treated with D,L-buthionine-[S,R]-sulphoximine to cause combined ascorbate and glutathione deficiency. The pool sizes and oxidation state of ascorbate and glutathione were not altered by deficiency of the other. alpha-Tocopherol and activities of monodehydroascorbate reductase, dehydroascorbate reductase, glutathione reductase, and catalase were little affected. Ascorbate peroxidase activity was higher in vtc1, vtc2-1, and vtc2-2. Ionically bound cell wall peroxidase activity was increased in vtc1, vtc2-1, and vtc4. Supplementation with ascorbate increased cell wall peroxidase activity. 2,6-Dichlorobenzonitrile, an inhibitor of cellulose synthesis, increased cell wall peroxidase activity in the wild type and vtc1. The transcript level of an endochitinase, PR1, and PR2, but not GST6, was increased in vtc1, vtc2-1, and vtc-2-2. Endochitinase transcript levels increased after ascorbate, paraquat, salicylic acid, and UV-C treatment, PR1 after salicylic acid treatment, and PR2 after paraquat and UV-C treatment. Camalexin was higher in vtc1 and the vtc2 alleles. Induction of PR genes, cell wall peroxidase activity, and camalexin in vtc1, vtc2-1, and vtc2-2 suggests that the mutants are affected in pathogen response signalling pathways.