This chapter considers the mechanistic value of determining the stereochemical consequence of an enzyme-catalyzed phosphoryl-transfer reaction and focuses on the methods for the synthesis and stereochemical analysis of chiral [18O, 17O, 18O] phosphoric monoesters. First chapter discusses the synthesis of [1(R)-16O, 17O, 18O] phospho-(S)-propane-1,2-dioP by the ephedrine route. P17OCl3, prepared from H217O and PCl5 in 85% yield, is allowed to react with (–)-ephedrine to yield the two diastereoisomeric chloro adducts in the approximate ratio 9: 1. Other method allows the stereospecific replacement of peripheral oxygen of a phosphoric diester either by sulfur or by oxygen. It is reported that the success of any stereospecific synthesis of chiral phosphoric esters is measured by its ability to yield a variety of esters of high isotopic purity that have a known configuration at phosphorus. To determine the absolute configuration of a labeled phosphoric monoester, one needs to define the absolute R or S sense of the peripheral oxygen isotopes of the phosphoryl group. The sense of isotopic substitution is determined in a diastereoisomer with respect to a separate chiral carbon center of known absolute configuration.