Abstract Changes in the concentrations of glycerate 3-phosphate, ribulose 1,5-bisphosphate, fructose 1,6-bisphosphate and dihydroxyacetone phosphate were measured in leaf discs under various conditions of light intensity and CO 2 concentration. The range of measured metabolite concentrations was then used to study metabolite regulation of ribulose-1,5-bisphosphate carboxylase. The effects of physiological concentrations of glycerate 3-phosphate, fructose 1,6-bisphosphate and dihydroxyacetone phosphate on ribulose-1,5-bisphosphate carboxylase activity of isolated stromal protein at saturating and limiting ribulose 1,5-bisphosphate were measured by incorporation of 14CO 2. A system containing ribulose 5-phosphate and an ATP-generating mechanism is described which was used to produce a constant low level of ribulose 1,5-bisphosphate in the reaction mixture. Of the metabolites measured glycerate 3-phosphate was the only one which was found to be present at a sufficiently high concentration relative to ribulose 1,5-bisphosphate in the light to be an effective inhibitor of ribulose-1,5-bisphosphate carboxylase activity in situ. Physiological concentrations of glycerate 3-phosphate were found to inhibit carboxylation severely, particularly at rate-limiting ribulose 1,5-bisphosphate concentrations. The rate of 14CO 2 assimilation was decreased 85% (with 0.2 mM ribulose 1,5-bisphosphate) by the presence of 20 mM glycerate 3-phosphate and the K m (ribulose 1,5-bisphosphate) of ribulose-1,5-bisphosphate carboxylase was increased by over 10-fold. The activity of the enzyme was inhibited substantially by a mixture of metabolites chosen to mimic in vivo conditions (20 mM glycerate 3-phosphate, 0.5 mM fructose 1,6-bisphosphate and 1.5 mM dihydroxyacetone phosphate). With this mixture of metabolites the K m (ribulose 1,5-bisphosphate) of ribulose-1,5-bisphosphate carboxylase was increased 10-fold above the control. A pulse of 10 mM glycerate 3-phosphate completely inhibited CO 2 uptake when the activity of the Benson-Calvin cycle as a whole was assayed in chloroplast extracts. Using these data we have examined the physiological significance of glycerate 3-phosphate modulation of ribulose-1,5-bisphosphate carboxylase at different metabolite levels.