Glycolysis is divided into two phases based on whether energy is utilized or released. While the first phase consumes ATP, the second phase produces energy in the form of ATP and NADH. The energy is released over a sequence of reactions that turns G3P into pyruvate. The energy-releasing phase—steps 6-10 of glycolysis—occurs twice, once for each of the two 3-carbon sugars produced during steps 1-5 of the first phase.

The first energy-releasing step—the 6th step of glycolysis —consists of two concurrent events: the oxidation and the phosphorylation of G3P. The electron carrier NAD⁺ removes one hydrogen atom from G3P, oxidizing the 3-carbon sugar and converting (reducing) NAD⁺ to form NADH and H⁺. The released energy is used to phosphorylate G3P, turning it into 1,3-bisphosphoglycerate.

Then, 1,3-bisphosphoglycerate is dephosphorylated to become 3-phosphoglycerate, donating the phosphate group to ADP to form ATP. The 3-phosphoglycerate is converted into an isomer, 2-phosphoglycerate.

2-phosphoglycerate loses a water molecule to become the unstable molecule 2-phosphoenolpyruvate or PEP. It quickly loses its phosphate group to ADP, creating a second ATP molecule and becoming pyruvate.

The energy-releasing phase releases two molecules of ATP and one molecule of NADH per converted sugar. Because it occurs twice—once for each 3-carbon sugar produced in the energy-requiring phase of glycolysis—four ATP molecules and two NADH molecules are released. In total, for each glucose molecule, glycolysis results in a net production of two ATP molecules (four ATPs produced and two ATPs consumed during the energy-requiring phase) and two NADH molecules.

Glycolysis produces two 3-carbon pyruvate molecules from one 6-carbon glucose molecule. In the presence of oxygen, pyruvate can be broken down into carbon dioxide in the Krebs cycle, releasing more ATP molecules. The NADH amasses in the cell, which can be converted back into NAD+ and used for further glycolysis.