Experiments with radioactive deoxycoformycin indicate that the inhibitor is released from calf intestinal adenosine deaminase after the enzyme-inhibitor complex is disrupted by denaturation. Experiments with 2H2O and H218O indicate that the enzyme does not catalyze elimination-addition reactions that could have led to reversible covalent derivatization of the enzyme. Ultraviolet difference spectra and the influence of pH on inhibitor binding indicate that deoxycoformycin is bound intact as the neutral species, at a binding site that is less polar than solvent water. The enzyme-inhibitor complex appears to be held together by hydrogen bonds of extraordinary stability (ca. 10 kcal/mol). These results suggest that deamination proceeds by direct water attack, the enzyme acting as a general-base catalyst.