Sulfur continues to be a valuable mechanistic probe for nucleophilic substitution at phosphorus. The stereochemical courses of many enzymatic phosphoryl- and nucleotidyltransferases have been elucidated by the use of P-chiral phosphorothio-analogs of biological substrates. The results have clarified the issue of single displacement versus double displacement mechanisms in enzyme catalysis. The principle of economy in the evolution of binding sites appears to govern whether an enzymatic phosphotransfer proceeds by a double displacement mechanism or a single displacement mechanism. The weakness of the P–S bond has allowed evidence for the transient formation of monomeric metaphosphate to be obtained in the hydrolysis of sym-μ-monothiopyrophosphate.