Acylphosphatase, one of the smallest enzymes, is expressed in all organisms; it displays hydrolytic activity on acyl phosphates, nucleoside di- and tri-phosphates, aryl phosphate monoesters, and polynucleotides, acyl phosphates being the most specific substrates in vitro. The mechanism of catalysis for human acylphosphatase (the organ common type isoenzyme) was investigated using both aryl phosphate monoesters and acyl phosphates as substrates. The enzyme is able to catalyze phosphotransfer from p-nitrophenyl phosphate to glycerol (but not from benzoyl phosphate to glycerol), as well as the inorganic phosphate-H218O oxygen exchange reaction in the absence of carboxylic acids or phenols. In short, our findings point to two different catalytic pathways for aryl phosphate monoesters and acyl phosphates. In particular, in the aryl phosphate monoester hydrolysis pathway, an enzyme-phosphate covalent intermediate is formed whereas the hydrolysis of acyl phosphate seems a more simple process in which the Michaelis-complex is directly attacked by a water molecule generating the reaction products. The formation of an enzyme-phosphate covalent complex is consistent with the experiments of isotope exchange and transphosphorylation from substrates to glycerol, as well as with the measurements of the Brønsted free energy relationships using a panel of aryl phosphates with different structures. His25 involvement in the formation of the enzyme-phosphate covalent complex during the hydrolysis of aryl phosphate monoesters finds significant confirmation in experiments performed with the His25Gln mutated enzyme.