The inactivation of S-adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) in isolated rat hepatocytes by 9-beta-D-arabinofuranosyladenine (ara-A) was associated with tight binding of ara-A to the enzyme and showed an initial phase obeying first-order kinetics characterized by Ki (concentration of half-maximal rate of inactivation) of 12 microM for ara-A and a maximal rate of inactivation of 0.7 min-1. Two to 3% of the enzyme in rat hepatocytes was not available for inactivation. Similar results were obtained with some cultured cells, including mouse plasmacytoma cells (MPC-11), mouse fibroblasts (L-929), and human chronic myelogenic leukemia cells (K-562). In a cellular medium devoid of adenosine deaminase, inhibitors of this enzyme did not affect the inactivation process in rat hepatocytes and only slightly enhanced this process in the cultured cells (at low concentrations of ara-A). Inactivation of AdoHcy hydrolase in rat hepatocytes was associated with a massive build-up of AdoHcy (from 75 to 5200 pmol/10(6) cells after 3 hr of incubation) and a moderate increase in cellular S-adenosylmethionine. The accumulation of AdoHcy in the cultured cells exposed to ara-A was less pronounced and no increase in cellular S-adenosylmethionine was observed. There was a quantitatively important export of AdoHcy from the rat hepatocytes and the cultured cells into the extracellular medium, whereas no leakage of S-adenosylmethionine was detected. The inactivation of AdoHcy hydrolase by ara-A in rat hepatocytes was inhibited in the presence of adenosine or homocysteine in the cellular medium. This effect of homocysteine correlated with increased cellular level of AdoHcy induced by this agent but was also associated with reduction in cellular uptake of ara-A.