Rubisco is responsible for the addition of the carbon atoms needed for the formation of carbohydrates, but by itself rubisco does not increase the amount of energy stored within the newly formed bonds. For this energy increase to take place, the carbon compounds formed by rubisco must be reduced. Nicotinamide adenine dinucleotide phosphate (NADPH) is the reducing agent used in the Calvin cycle. NADPH transfers the electrons that allow carbohydrates to be synthesized from CO₂ (Fig. 8.5).

Like all components of the Calvin cycle, NADPH can move freely within the stroma of the chloroplast. Although NADPH is a powerful reducing agent, energy and electrons are transferred from NADPH only under the catalysis of a specific enzyme, thus providing a high degree of control over the fate of these electrons. In the Calvin cycle, the reduction of 3-PGA involves two steps: (1) ATP donates a phosphate group to 3-PGA, and (2) NADPH transfers two electrons plus one proton (H+) to the phosphorylated compound, which releases one phosphate group (P_i). Because two molecules of 3-PGA are formed each time rubisco catalyzes the incorporation of one molecule of CO₂, two ATP and two NADPH are required for each molecule of CO₂ incorporated by rubisco. NADPH provides most of the energy incorporated in the bonds of the carbohydrate molecules produced by the Calvin cycle. Nevertheless, ATP plays an essential role in preparing 3-PGA for the addition of energy and electrons from NADPH.

These energy transfer steps result in the formation of 3-carbon carbohydrate molecules known as triose phosphates. Triose phosphates are the true products of the Calvin cycle and they are the principal form in which carbohydrates are exported from the chloroplast during photosynthesis. Larger sugars, such as glucose and sucrose, are assembled from triose phosphates in the cytoplasm.

If every triose phosphate molecule produced by the Calvin cycle were exported from the chloroplast, RuBP could not be regenerated and the Calvin cycle would grind to a halt. In fact, most of the triose phosphate molecules must be used to regenerate RuBP. For every six triose phosphate molecules that are produced, only one can be withdrawn from the Calvin cycle.