Binding of adenosine to S-adenosyl-L-homocysteine (AdoHcy) hydrolase (EC 3.3.1.1.) and partial conversion of bound adenosine to a substance liberating adenine has been demonstrated under conditions of enzymatic synthesis and hydrolysis of ADoHcy (Ueland, P. M., and Helland, S. (1980) J. Biol. Chem. 255, 7722-7727). Gel filtration of cytosol from isolated rat hepatocytes treated with [14C]adenosine on a high performance liquid chromatography protein column showed that labeled adenine/adenosine eluted as a peak which co-chromatographed exactly with AdoHcy hydrolase. Formation of this peak was inhibited by exposure of the cells to compounds (ara-A, 3-deazaadenosine, and homocysteine) interacting with the catalytic site of the enzyme. Furthermore, the adenine/adenosine-protein complex and AdoHcy hydrolase focused at exactly the same pH (pI = 5.76) in a granulated bed. On this basis it was concluded that labeled adenosine formed a stable complex with AdoHcy hydrolase. A substantial portion (about 50%) of endogenous adenosine in rat hepatocytes seemed to be associated with AdoHcy hydrolase, and this portion equaled the amount of cellular adenosine which was not readily mobilized by high level of extracellular adenosine deaminase. Exposure of the hepatocytes to compounds which block the formation of the adenosine-AdoHcy hydrolase complex (ara-A, 3-deazaadenosine, and homocysteine) for 1 to 2.5 h only slightly reduced the amount of adenosine associated with the enzyme, indicating a slow turnover of the complex under the conditions of the experiment. It was concluded that adenosine is sequestered in rat hepatocytes through the interaction with AdoHcy hydrolase. The physiological implication of this process may be related to the metabolism and biological effects of adenosine as well as the regulation of AdoHcy hydrolase activity.