Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of the P-O 5′ bond in RNA. Although this enzyme has been the object of much seminal work in biological chemistry, the nature of its rate-limiting transition state and its catalytic rate enhancement had been unknown. Here, the value of k cat /K m for the cleavage of UpA by wild-type RNase A was found to be inversely related to the concentration of added glycerol. In contrast, the values of k cat /K m for the cleavage of UpA by a sluggish mutant of RNase A and the cleavage of the poor substrate UpOC 6 H 4 -p-NO 2 by wild-type RNase A were found to be independent of glycerol concentration. Yet, the values of k cat /K m for UpA cleavage by the wild-type and mutant enzymes were found to have an identical dependence on the concentration of added sucrose. Although both glycerol and sucrose are viscogenic, only glycerol interacts strongly with single-stranded nucleic acids. Catalysis of UpA cleavage by RNase A is therefore limited by substrate desolvation. The rate of UpA cleavage by RNase A is maximal at pH 6.0, where k cat = 1.4 × 10 3 s −1 and k cat /K m = 2.3 × 10 6 M −1 s −1 at 25°C. At pH 6.0 and 25°C, the uncatalyzed rate of [5,6- 3 H]Up[3,5,8- 3 H]A cleavage was found to be k uncat = 5 × 10 −9 s −1 (t 12 = 4 years). Thus, RNase A enhances the rate of UpA cleavage by 3 × 10 11 -fold by binding to the transition state for P-O 5′ bond cleavage with a dissociation constant of <2 × 10 −15 M.