Kinetic isotope effects have been measured for the aqueous hydrolysis reactions of p-nitrophenyl phosphorothioate (pNPPT) and the diester ethyl p-nitrophenyl phosphorothioate, and for the alkaline phosphatase-catalyzed reaction with pNPPT. The results show that the transition states of the uncatalyzed reactions of the phosphorothioate mono- and diesters are very similar to those of the corresponding phosphate ester reactions. The secondary ¹⁸O nonbridge isotope effects in reactions of phosphate esters become more normal as the mechanism changes from dissociative, metaphosphate-like to associative, phosphorane-like. The opposite trend occurs in phosphorothioate esters, due to differences in the relative contributions of bond-order changes and bending modes to this isotope effect. The KIEs for the alkaline phosphatase-catalyzed reaction of pNPPT are most consistent with a tight, triester-like transition state, probably a result of perturbations resulting from the larger size of sulfur that lead to a nucleophile attack angle that is unfavorable for an in-line process with a loose transition state.