Post-translational modifications have major importance for the structure and function of a multiplicity of proteins. Phosphorylation is a widespread phenomenon among eukaryotic proteins. Whereas O-phosphorylation on the side chains of serine, threonine, and tyrosine in proteins is well known and has been studied extensively, to our knowledge the endogenous phosphorylation of hydroxyproline has not previously been reported. In the present work, we provide evidence for the first time that O-phosphohydroxyproline (Hyp(P)) is a proteinogenic amino acid. To detect Hyp(P) in proteins we generated a Hyp(P)-specific polyclonal antibody. We could identify Hyp(P) in various proteins by Western blot analysis, and we characterized the sequence position of Hyp(P) in the protein α-crystallin A by electrospray ionization-tandem mass spectrometry. Our experiments clearly demonstrate hydroxylation and subsequent phosphorylation of a proline residue in α-crystallin A in the eye as well as in heart tissue of rat.