There is currently considerable interest in the prospects for bioengineering crassulacean acid metabolism (CAM) photosynthesisor key elements associated with it, such as increased water-use efficiencyinto C 3 plants. Resolving how CAM photosynthesis evolved from the ancestral C 3 pathway could provide valuable insights into the targets for such bioengineering efforts. It has been proposed that the ability to accumulate organic acids at night may be common among C 3 plants, and that the transition to CAM might simply require enhancement of pre-existing fluxes, without the need for changes in circadian or diurnal regulation. We show, in a survey encompassing 40 families of vascular plants, that nocturnal acidification is a feature entirely restricted to CAM species. Although many C 3 species can synthesize malate during the light period, we argue that the switch to night-time malic acid accumulation requires a fundamental metabolic reprogramming that couples glycolytic breakdown of storage carbohydrate to the process of net dark CO 2 fixation. This central element of the CAM pathway, even when expressed at a low level, represents a biochemical capability not seen in C 3 plants, and so is better regarded as a discrete evolutionary innovation than as part of a metabolic continuum between C 3 and CAM.