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This paper originates from a presentation at the IIIrd International Congress on Crassulacean Acid Metabolism, Cape Tribulation, Queensland, Australia, August 2001. In crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) kinase is expressed at night under the control of a circadian oscillator. We have proposed that this is an indirect effect, secondary to circadian fluctuations in the level of a metabolite, possibly cytosolic malate, resulting from a primary effect on the permeability of the tonoplast (Nimmo 2000, Trends in Plant Science 5 , 75-80). Here we show that the nocturnal accumulation of PEPC kinase translatable mRNA and phosphorylation of PEPC in Kalanchoë fedtschenkoi is blocked by the protein phosphatase inhibitor cantharidin. This implicates protein dephosphorylation in the circadian pathway that regulates expression of PEPC kinase. We also show that the effect of reducing the temperature from 30 to 15 °C on CO2 fixation by detached leaves held in constant darkness and normal air is 'gated' by the circadian clock. This strongly supports the view that the effect of the clock on the expression of PEPC kinase is secondary rather than direct. We have developed a non-aqueous fractionation protocol that separates the cytosolic material in mature leaves from vacuolar material. The cytosolic malate in mature leaves represents a very small part of the total malate, and its concentration cannot be measured precisely by this method.