In the presence of Mg²⁺ ions, inositol monophosphatase from bovine brain catalyses the hydrolysis of the phosphate ester of a range of purine- and pyrimidine-containing nucleoside 2′-phosphates including adenosine 2′-phosphate (2′-AMP) but not adenosine 2′-phosphorothioate (2′-AMPS). 2′-AMPS also fails to serve as an inhibitor under these conditions. In contrast to the situation for the alcohol hydrolysis product, inositol, adenosine does not serve as a product inhibitor for the enzyme or mediate the enzyme-catalysed exchange of ¹⁸O-label from [¹⁸O]water into inorganic phosphate. However, in the presence of Mn²⁺ ions 2′-AMPS is a substrate for the enzyme. These findings indicate that the product adenosine does not bind to the enzyme in its ground-state conformations and that a strong phosphate group–holoenzyme interaction is required to stabilise a high-energy arrangement in the enzyme–substrate complexes of 2′-AMPS and, probably, 2′-AMP. On the basis of these results and those from previous kinetic and substrate modification studies it is proposed that a second Mg²⁺ ion might stabilise a conformation in which the adenine moiety of bound 2′-AMP occupies a C-1′-axial ribofuranosyl position through the direct chelation of the second Mg²⁺ ion to the bridging phosphate ester 2′-O-atom and the ribofuranose ring O-atom. An alternative high-energy arrangement in which the interaction of the second Mg²⁺ ion with the ribofuranose ring O-atom is mediated via water, such that the conformational strain in the furanose ring is relaxed, but where the entropy of the water is decreased, is also a possibility.