Abstract Crystalline ribulose 1,5-bisphosphate carboxylase-oxygenase (EC 126.96.36.199) was isolated from tobacco ( Nicotiana tabacum L.) leaf homogenates and the two competing reactions were examined for differential regulation in vitro by temperature pretreatment and chloroplast metabolites. Both the carboxylase and oxygenase activities were inactivated 50% by storing the dissolved protein at 0 °C and fully reactivated by heating the solution at 50 °C in the absence of Mg 2+ and a sulfhydryl reagent. When the heat-activated enzyme was preincubated with physiological levels of various chloroplast metabolites and CO 2 and the two reactions were assayed simultaneously in the same reaction vessel upon initiation with ribulose 1,5-bisphosphate, three classes of effectors were observed: (a) those which stimulated both activities (NADPH, 6-phosphogluco-bisphosphate gluconate, fructose 1,6-bisphosphate, 3-phosphoglycerate glycerate), (b) those which essentially had no effect (fructose 6-phosphate, glucose 6-phosphate), and (c) one, ribose 5-phosphate, which inhibited the two reactions. However, within the limits of experimental error, none of the metabolites examined produced a differential regulation of the ribulose 1,5-bisphosphate carboxylase-oxygenase activities. The similar response of the two competing activities to temperature pretreatment and various chloroplast metabolites is consistent with the notion that both reactions are associated with the same or adjacent catalytic sites on this bifunctional enzyme.