Isolation of bovine S-adenosylhomocysteine hydrolase by affinity chromatography unexpectedly yielded two forms of the enzyme. Type A contained 4 moles of NAD\sp+/mole of enzyme tetramer, while Type B had only half that number of nucleotide cofactors associated with it. The acetylenic analog of adenosine, 9-(5\sp′,6\sp′-dideoxy-β-D-ribo-hex-5\sp′-ynofuranosyl)-adenine, was synthesized, and its behavior as an inhibitor of Type A enzyme examined. Irreversible inactivation of the enzyme with excess inhibitor was accompanied by the reduction of 2 equivalents of NAD\sp+ to NADH, release of the remaining NAD\sp+ from the enzyme, and binding of 4 equivalents of inhibitor per enzyme tetramer. Denaturation studies suggested that two equivalents of the inhibitor may form a covalent bond between the enzyme and the oxidized inhibitor. The inactivation behavior exhibited by Type A and Type B enzyme with 2\sp′-deoxy-2\sp′,2\sp′-difluoroadenosine was compared. The Type B enzyme was irreversibly inactivated by excess inhibitor with concurrent reduction of 1 mole of NAD\sp+ to NADH and release of 1 mole of NAD\sp+. Inactivation of the Type A enzyme was accompanied by the reduction of 1 mole of NAD\sp+ to NADH and release of ca. 2 moles of NAD\sp+. Upon dialysis, 50% of the original activity of the enzyme was regained. Binding stoichiometries of 1 and 3 moles inhibitor/mole of enzyme tetramer were determined for the Type B and Type A enzyme, respectively. Denaturation studies indicated that 1 equivalent of the oxidized inhibitor may have formed a covalent bond with the Type A enzyme, but not with Type B. The binding behavior of adenosine with both types of the enzyme was also studied. Type B enzyme exhibited a large binding affinity of 4-25 moles of adenosine/mole of enzyme tetramer, while Type A bound only 3 moles of adenosine/mole enzyme tetramer. The binding of adenosine to the Type B enzyme was accompanied by the reduction of 1 equivalent of NAD\sp+ to NADH. Denaturation experiments suggested the formation of a covalent bond between the enzyme and one equivalent of adenosine. However, peptide mapping of the reduced enzyme-adenosine complex failed.