In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k up-regulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C4 protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH4Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP3)-gated tonoplast calcium channels, and a putative Ca2+/calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C4 (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis.