Vitamin K (2-methyl-3-phytyl-1,4-naphthoquinone) is required for the synthesis of prothrombin, Factor VII, Factor IX, Factor X, and a number of newly discovered proteins. These plasma proteins participate in calcium-dependent phospholipid membrane interactions which are mediated through the presence of gamma-carboxyglutamyl residues in their amino-terminal region. Vitamin K is required for the postribosomal conversion of glutamyl residues in liver precursors of these proteins to gamma-carboxyglutamyl residues in the completed plasma proteins. In the absence of vitamin K, or in the presence of vitamin K antagonists, animals produce plasma forms which lack the carboxylated residue. These proteins are nonfunctional because of their lack of phospholipid interaction. The vitamin K-dependent carboxylase which carries out this reaction has been studied in rat liver microsomal preparations where it will carboxylate the endogenous precursor proteins. Low-molecular-weight glutamyl-containing peptide substrates, such as Phe-Leu-Glu-Glu-Leu, which are homologous to regions of the prothrombin precursor, will also serve as substrates for the detergent-solubilized enzyme. This enzyme has been shown to require the reduced form of the vitamin and O2 but no ATP or a biotin-containing protein for its activity. The same microsomal preparations will also convert vitamin K to its 2,3-epoxide, and it is possible that activity may be related to the role of the vitamin in driving the carboxylase reaction.