The properties of the methylphosphonyl group as a substrate analog for the phosphoryl moiety of various biol. phosphoryl donors was investigated in several enzymic phosphoryl transfer reactions.The synthesis and characterization of adenosine 5'-(β-methylphosphonyl)diphosphate, adenosine 5'-methylphosphonate, acetyl methylphosphonate, and methylphosphonoenolpyruvate are fully described.Adenosine 5'-(β-methylphosphonyl)diphosphate is not a substrate for adenylate kinase, hexokinase, 3-phosphoglycerate kinase, glycerol kinase, phosphofructokinase, creatine kinase, alk. phosphatase, or nucleoside 5'-diphosphophate kinase.Competitive inhibition of ATP was observed with hexokinase and 3-phosphoglycerate kinase with Ki/Km ≃10.Adenosine 5'-methylphosphonate was a substrate for adenylate deaminase and 5'-nucleotidase, but not for adenylate kinase, acid phosphatase, 5'-phosphodiesterase, or 3'-phosphodiesterase.Acetyl methylphosphonate inhibits the reaction of acetyl phosphate with acetate kinase, but methylphosphonoenolpyruvate has no effect upon the reaction of phosphoenolpyruvate with pyruvate kinase.The results indicate that with the exception of 5'-nucleotidase, the methylphosphonyl group is incapable of undergoing transfer.One interpretation among others is that a metaphosphate-type mechanism is required for these processes.