In  plants, the first step in the light-independent reactions of photosynthesis is the fixation of  by the enzyme RuBisCO to form 3-phosphoglycerate. However, RuBisCo has a dual carboxylase and oxygenase activity. Oxygenation results in part of the substrate being oxidized rather than carboxylated, resulting in loss of substrate and consumption of energy, in what is known as photorespiration. Oxygenation and carboxylation are competitive, meaning that the rate of the reactions depends on the relative concentration of oxygen and .
In order to reduce the rate of photorespiration,  plants increase the concentration of  around RuBisCO. To do so two partially isolated compartments differentiate within leaves, the mesophyll and the bundle sheath. Instead of direct fixation by RuBisCO,  is initially incorporated into a four-carbon organic acid (either malate or aspartate) in the mesophyll. The organic acids then diffuse through plasmodesmata into the bundle sheath cells.  There, they are decarboxylated creating a -rich environment. The chloroplasts of the bundle sheath cells convert this  into carbohydrates by the conventional  pathway.
There is large variability in the biochemical features of C4 assimilation, and it is generally grouped in three subtypes, differentiated by the main enzyme used for decarboxylation ( NADP-malic enzyme, NADP-ME; NAD-malic enzyme, NAD-ME; and PEP carboxykinase, PEPCK). Since PEPCK is often recruited atop NADP-ME or NAD-ME it was proposed to classify the biochemical variability in two subtypes. For instance, maize and sugarcane use a combination of NADP-ME and PEPCK, millet uses preferentially NAD-ME and Megathyrsus maximus, uses preferentially PEPCK.