Hydrolytic reactions of two trinucleoside 3',3',5'-monophosphates (1a and 1b) have been followed by RP HPLC over a wide pH range to elucidate the role of the 2'-OH group of the departing 3'-linked nucleoside as an intramolecular hydrogen bond donor accelerating the cleavage. In aqueous alkali la is cleaved 27 times as rapidly as 1b. The product distribution, however, remains unchanged upon methylation of the 2'-oxygen: P-O3' bond cleavage accounts for 88% of the total reaction with both model phosphotriesters. The results suggest that the phosphorane intermediate, rather than the departing 3'-oxyanion, is stabilized by intramolecular hydrogen bonding.