Vitamin K is the blood-clotting vitamin. It participates in the blood coagulation cascade as a carboxylase cofactor. Enzymic oxygenation of vitamin K hydroquinone provides the driving force for the carboxylation of selected glutamates in the proteins of the blood-clotting cascade. The active site of vitamin K has now been defined by O-18-labeling experiments. The oxygenation is completely specific for the carbonyl group adjacent to the quinone methyl group of vitamin K. The experiment makes use of the O-18-labeled vitamin K isotopomers 9 and 10. Thus, oxygenation of 9 with O-16(2) occurs at the carbonyl group next to methyl, as shown by exchange of the O-18 label at that position. Synthesis of the two O-18-labeled vitamin K isotopomers 9 and 10 was accomplished by cerium(IV)-mediated oxidation in the presence of (H2O)-O-18 of the corresponding methyl half-ethers 4 and 8. The position of the label was ascertained by C-13 and heteronuclear NOE NMR spectroscopies. A role for the active site thiols on the vitamin K-dependent carboxylase is also suggested. The thiolate anion is an excellent candidate for the weak base that initiates the base strength amplification sequence leading to carboxylation and vitamin K oxide formation.