Leaf photosynthesis and ribulose bisphosphate carboxylase activation level were inhibited in several mutants of the C(3) crucifer Arabidopsis thaliana which possess lesions in the photorespiratory pathway. This inhibition occurred when leaves were illuminated under a photorespiratory atmosphere (50% O(2), 350 microliters per liter CO(2), balance N(2)), but not in nonphotorespiratory conditions (2% O(2), 350 microliters per liter CO(2), balance N(2)). Inhibition of carboxylase activation level was observed in strains with deficient glycine decarboxylase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, glutamate synthase, and chloroplast dicarboxylate transport activities, but inhibition did not occur in a glycolate-P phosphatase-deficient strain. Also, the photorespiration inhibitor aminoacetonitrile produced a decline in leaf and protoplast ribulose bisphosphate carboxylase activation level, but was without effect on intact chloroplasts. Fructose bisphosphatase, a light-activated enzyme which is strongly dependent on stromal pH and Mg(2+) for regulation, was unaffected by conditions which caused inhibition of ribulose bisphosphate carboxylase. Thus, the mechanism of inhibition does not appear to involve changes in stromal Mg(2+) and pH but rather is associated with metabolite flux through the photorespiratory pathway.