Heavy-atom isotope effects have been used to characterize the transition states for the aqueous hydrolysis reactions of the p-nitrophenyl phosphate dianion and monoanion, for the reaction of the dianion in neat tert-butyl alcohol, and for the reaction catalyzed by alkaline phosphatase. The primary oxygen-18 isotope effect at the phenolic oxygen ((18)k(bridge)), the secondary nitrogen-15 effect ((15)k) in the nitrogen atom of the leaving group, and the secondary oxygen-18 isotope effects in the nonbridge oxygen atoms of the phosphoryl group ((18)k(nonbridge)) have been measured. The isotope effects for the dianion reaction in water at 95 degrees C were (15)k = 1.0028 +/- 0.0002, (18)k(bridge) = 1.0189 +/- 0.0005, and (18)k(nonbridge) = 0.9994 +/- 0.0005. The dianion reaction in tert-butyl alcohol at 30 degrees C gave values of (15)k = 1.0039 +/- 0.0003, (18)k(bridge) = 1.0202 +/- 0.0008, and (18)k(nonbridge) = 0.9997 +/- 0.0016. When corrected for temperature, the results are very similar, indicating similar late transition state structures for the two reactions with little or no change in bond order between the phosphorus and the nonbridge oxygen atoms. The isotope effects on the aqueous reaction of the monoanion were (15)k = 1.0004 +/- 0.0002, (18)k(bridge) = 1.0087 +/- 0.0003, and (18)k(nonbridge) = 1.0184 +/- 0.0005, suggesting both proton transfer and bond cleavage are rate-limiting. The isotope effects on the alkaline phosphatase reaction are all near unity, indicating that a nonchemical step is rate-limiting for the enzymatic reaction.