Hydrolytic reactions of 2′,3′-O-methyleneadenos-5′-yl 2′,5′-di-O-methylurid-3′-yl 5′-O-methylurid-3′(2′)-yl phosphate (1a,b) have been followed by RP-HPLC over a wide pH range to evaluate the feasibility of occurrence of phosphate-branched RNA under physiological conditions. At pH <2, where the decomposition of 1a,b is first order in [H₃O⁺], the P–O5′ bond is cleaved 1.5 times as rapidly as the P–O3′ bond. Under these conditions, the reaction probably proceeds by an attack of the 2′-OH on the phosphotriester monocation. Over a relatively wide range from pH 2 to 5, the hydrolysis is pH-independent, referring to rapid initial deprotonation of the attacking 2′-OH followed by general acid catalyzed departure of the leaving nucleoside. The P–O5′ bond is cleaved 3 times as rapidly as the P–O3′ bond. At pH 6, the reaction becomes first order in [HO⁻], consistent with an attack of the 2′-oxyanion on neutral phosphate. The product distribution is gradually inversed: in 10 mmol L⁻¹ aqueous sodium hydroxide, cleavage of the P–O3′ bond is favored over P–O5′ by a factor of 7.3. The results of the present study suggest that the half-life for the cleavage of 1a,b under physiological conditions is only 100 s. Even at pH 2, where 1a,b is most stable, the half-life for its cleavage is less than one hour and the isomerization between 1a and 1b is even more rapid than cleavage. The mechanisms of the partial reactions are discussed.