The S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) activity and the metabolism of AdoHcy were investigated in various tissues of mice given a single injection or repetitive injections of 9-beta-D-arabinofuranosyladenine (ara-A) with and without the adenosine deaminase inhibitor, 2'-deoxycoformycin (dCF). A single injection of ara-A (50 mg/kg) rapidly inactivated AdoHcy hydrolase in several organs (liver, kidney, spleen, lung, heart, skeletal muscle, and brain). Then, the enzyme activity in these tissues gradually recovered. This process, termed reactivation of AdoHcy hydrolase, was not sensitive to cycloheximide but was partly inhibited by dCF. In the absence of dCF, nearly no increase in AdoHcy content in the tissues was observed, whereas a single injection of ara-A plus dCF induced a small, transient increase in AdoHcy content of most tissues. Repetitive injections of ara-A (without dCF) caused a moderate increase in the AdoHcy level of tissues, whereas repetitive injections of the drug combination ara-A plus dCF resulted in a massive accumulation of AdoHcy in liver and kidney and, to a lesser degree, in other tissues. A moderate increase in S-adenosyl-L-methionine was observed in some tissues. These metabolic effects were associated with a rapid inactivation of AdoHcy hydrolase, but a fraction of the enzyme activity (about 8% in liver) was not or only slowly inactivated. AdoHcy accumulated in serum of mice receiving this treatment. Treatment of mice with dCF alone for up to 10 hr induced no increase in AdoHcy content of the tissues.