The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase displayed near-maximal activity in isolated, intact barley (Hordeum vulgare L. cv. Pennrad) mesophyll protoplasts. The carboxylase deactivated 40 to 50% in situ when protoplasts were dark-incubated 20 minutes in air-equilibrated solutions. Enzyme activity was fully restored after 1 to 2 minutes of light. Addition of 5 millimolar NaHCO(3) to the incubation medium prevented dark-inactivation of the carboxylase. There was no permanent CO(2)-dependent activation of the protoplast carboxylase either in light or dark. Activation of the carboxylase from ruptured protoplasts was not increased significantly by in vitro preincubation with CO(2) and Mg(2+). In contrast to the enzyme in protoplasts, the carboxylase in intact barley chloroplasts was not fully reactivated by light at atmospheric CO(2) levels. The lag phase in carbon assimilation was not lengthened by dark-adapting protoplasts to low CO(2) demonstrating that light-activation of the carboxylase was not involved in photosynthetic induction. Irradiance response curves for reactivation of the the carboxylase and for CO(2) fixation by isolated barley protoplasts were similar. The above results show that there was a fully reversible light-activation of the carboxylase in isolated barley protoplasts at physiologically significant CO(2) levels.