Before the pyruvates from glycolysis (see Fig. 2) can enter the citric acid cycle, they must undergo a transition reaction. The 3-carbon pyruvate is converted into a 2-carbon acetyl group with a carboxyl being removed as CO₂. The acetyl group is attached to coenzyme A to form acetyl coenzyme A (acetyl-CoA), a key precursor metabolite. As the two acetyl groups become oxidized to acetyl-CoA, two molecules of NAD⁺ are reduced to 2NADH + 2H⁺.

The two molecules of acetyl-CoA then enter the citric acid cycle (see Fig. 17). The 2NADH molecules that are produced carry electrons to the electron transport system for further production of ATPs by oxidative phosphorylation (see Fig. 28).