This chapter discusses the biological hydroxylations and ascorbic acid with regard to collagen metabolism. Collagen hydroxyproline and hydroxylysine are formed by the enzymatic hydroxylation of specific proline and lysine residues incorporated into peptide linkage. When, in isolated collagen-forming systems, hydroxylation is prevented by the exclusion of oxygen or by means of the chelating agent, α,α′-dipyridyl, an unhydroxylated polypeptide termed protocollagen is produced. Separate enzymes, known as protocollagen proline and lysine hydroxylase, are involved in the formation of collagen hydroxyproline and hydroxylysine. They belong to a class of hydroxylases, members of which all require for activity, molecular oxygen, ferrous ion, a 2-ketoacid, and a reductant, the most effective being ascorbic acid. The α-ketoacid is the cosubstrate in the mixed function oxidation, and undergoes a stoichiometric decarboxylation during the course of the hydroxylation. The reduction in hydroxylation is regarded as direct evidence for the participation of ascorbic acid in the hydroxylation of protocollagen proline in vivo. The occurrence in ascorbic acid deficiency of a normal or even increased hydroxyproline excretion is not immediately reconcilable with inhibited hydroxylation.