The diel (24-h) Crassulacean acid metabolism (CAM) cycle in Mesembryanthemum crystallinum (L.) requires rhythmic patterns of transitory starch degradation to produce carbon skeletons for phosphoenolpyruvate (PEP) synthesis during the nocturnal Phase I, when PEP carboxylase (PEPc) mediates CO2 fixation. Under a normal light-dark cycle, nocturnal malate accumulation and nocturnal CO2 uptake were observed for CAM-induced, but not C-3, M. crystallinum. In both C3 and CAM plants, transcripts encoding beta-amylase and starch phosphorylase accumulated during the afternoon and declined nocturnally. Under a continuous light regime, ribulose-1,5-bisphosphate carboxylase/oxygenase activity remained co-ordinated with the CAM phases, and circadian abundance patterns were observed for transcripts encoding starch degradative enzymes. Despite circadian PEPc kinase expression, the accumulation of vacuolar malate ceased under continuous light. Exposure to CO2-free air for 24 h inhibited starch accumulation over the photoperiod, but re-fixation of respiratory CO2 resulted in the overnight accumulation of malate to levels comparable to those of control plants. Upon return to normal air, the depleted starch concentration led to stoichiometric decreases in Phase-I CO2 uptake and malate accumulation. The up-regulation of PEPc kinase transcripts under these conditions was ineffective at sustaining Phase-I CO2 uptake under starch-limited conditions. We conclude that starch turnover regulates and limits carbon flux through the diel CAM cycle.