The mechanism of methanol photorelease from 2-nitrobenzyl methyl ether (1) and 1-(2-nitrophenyl)ethyl methyl ether (2), and of ATP release from adenosine-5'-triphosphate-[P-3-(1-(2-nitrophenyl)ethyl)] ester ('caged ATP', 3) was studied in various solvents by laser flash photolysis with UV-vis and IR detection. In addition to the well-known primary aci-nitro transients (A, lambda(max) approximate to 400 nm), two further intermediates preceding the release of methanol, namely the corresponding 1,3-dihydrobenz[c]isoxazol-1-ol derivatives (B) and 2-nitrosobenzyl hemiacetals (C), were identified. The dependencies of the reaction rates of A-C on pH and buffer concentrations in aqueous solution were studied in detail. Substantial revision of previously proposed reaction mechanisms for substrate release from 2-nitrobenzyl protecting groups is required: (a) A novel reaction pathway of the aci-tautomers A prevailing in buffered aqueous solutions, e.g., phosphate buffer with pH 7, was found. (b) The cyclic intermediates B were identified for the first time as the products formed by the decay of the aci-tautomers A in solution. A recently proposed reaction pathway bypassing intermediates B (Corrie et al. J. Am. Chem. Soc., 2003, 125, 8546-8554) is shown not to be operative. (c) Hemiacetals C limit the release rate of both 1 (pH < 8) and 2 (pH < 10). This observation is in contrast to a recent claim for related 2-nitrobenzyl methyl ethers (Corrie et al.). Our findings are important for potential applications of the 2-nitrobenzyl protecting group in the determination of physiological response times to bioagents ('caged Compounds').