Using gas exchange, enzyme assays, and theoretical modeling of photosynthetic responses to light and CO(2), we investigated whether decarbamylation of the active site of Rubisco at low CO(2) and low light leads to a condition where the activation state of Rubisco directly limits the rate of net CO(2) assimilation. Photosynthetic limitation by a reduction in the activation state of Rubisco would be indicated as a decline in the initial slope of the photosynthetic CO(2) response relative to what is predicted using theoretical models. In bean (Phaseolus vulgaris) and oat (Avena sativa), we saw no discrepancy between predicted and observed initial slope values at 200 and 400 mbar O(2), indicating no limitation by the carbamylation state of Rubisco. At 30 mbar O(2) and light saturation, we also saw no discrepancy between predicted and observed initial slope values; however, at subsaturating light intensity, our observed initial slope values were less than the modeled initial slope values that corresponded to an RuBP regeneration limitation. Moreover, significant reduction of the Rubisco activation state occurred in both species at 30 mbar O(2) and 30 mubar CO(2). When the model was reprogrammed to account for observed levels of Rubisco deactivation, the predicted and measured initial slope values at low O(2) and low PPFD were similar, indicating the reduction in carbamylation state accounted for the discrepancy. We interpret this as evidence for a direct limitation of the carbamylation state of Rubisco, probably because of a CO(2) limitation for carbamate formation. This limitation was only observed at intercellular CO(2) levels below what is encountered in vivo. At physiologically relevant CO(2) levels in situ, the leaves maintained sufficient Rubisco activity to avoid cabamylation state limitations in the steady state.