The intracellular locations of six key enzymes of Crassulacean acid metabolism were determined using enzymically isolated mesophyll protoplasts of Sedum praealtum D.C. Data from isopycnic sucrose density gradient centrifugation established the chloroplastic location of pyruvate Pi dikinase, the mitochondrial location of NAD-linked malic enzyme, and exclusively nonparticulate (not associated with chloroplasts, peroxisomes, or mitochondria) locations of phosphoenolpyruvate carboxylase, NADP-linked malic enzyme, enolase, and phosphoglycerate mutase. The consequences of this enzyme distribution with respect to compartmentalization of the pathway and the transport of metabolites in Crassulacean acid metabolism are discussed.