In biochemistry, phosphorylation is the addition of a phosphate (PO4) group to a protein or a small molecule. Phosphorylation is a tremendously important event. In the database MedLine almost a hundred thousand articles on phosphorylation are present. The large majority of these is dedicated to protein phosphorylation.

Beyond doubt, protein phosphorylation is the most important regulatory event in eukaryotic cells. Many enzymes and receptors are turned on or off by phosphorylation and dephosphorylation. Phosphorylation is catalyzed by various specific protein kinases, whereas phosphatases dephosphorylate. Regulation of protein activity is very important in cells. For example, the p53 tumor suppressor gene activates genes that cause a cell to stop growing, or even to kill itself (apoptosis). However, this activity should only be present if the cell is damaged. Therefore, the p53 protein is extensively regulated. In fact, p53 contains more than 18 phosphorylation sites.

Phosphorylation is a very fast way of regulating proteins. In the simplest way of regulation, the protein is simply not there until it is needed. Steroid hormones like estrogen, for example, act as transcription factors, causing the proteins they regulate to be produced. However, this takes time, and it also takes time until the proteins degrade again and the action stops. If the protein is regulated by phosphorylation, it is constantly present in "standby" mode. When an activating signal arrives, the protein becomes phosphorylated and performs its action. Upon the deactivating signal, the protein becomes dephosphorylated again and stops working. This is the mechanism in many forms of signal transduction, for example the way in which incoming light is processed in the light-sensitive cells of the retina. The network underlying phosphorylation can be very complex. Often, protein A phosphorylates B, and B phosphorylates C, but a also phosphorylates C directly, and B can phosphorylate D, which may in turn phosphorylate A.

Within a protein, phosphorylation can occur on several amino acids. Phosphorylation on serine is the most common, followed by threonine. Tyrosine phosphorylation is the most rare. However, since tyrosine phosphorylated proteins are relatively easy to purify using antibodies, tyrosine phosphorylation sites are relatively well understood.

There are other kinds of phosphorylation besides protein phosphorylation:

ATP, the "high-energy" exchange medium in the cell, is synthesized in the mitochondrion by addition of a third phosphate group to ADP in a process referred to as oxidative phosphorylation. ATP is then used at various points in the series of reactions that constitute glycolysis, to transfer energy to other small molecules.

ATP is synthesized at the expense of solar energy by photophosphorylation in the chloroplasts of plant cells.