Most S-adenosylmethionine (AdoMet)-dependent methyltransferases are regulated in vivo by the AdoMet/S-adenosylhomocysteine (AdoHcy) ratio, also termed as “methylation potential.” Since adenosine inhibits in vitro AdoHcy hydrolysis and since adenosine tissue levels increase during hypoxia, it can be predicted that AdoHcy levels may increase in the rat kidney in parallel of those of adenosine. Therefore, the present investigation was performed to assess changes of renal AdoHcy and AdoMet tissue contents during ischemia and after administration of adenosine and homocysteine or both in the ischemic rat kidney. In anesthetized rats ischemia of the kidney was induced by renal artery occlusion for various time intervals. Adenosine and homocysteine were infused into the renal artery of the ischemic kidney. To induce a hyperhomocysteinemia homocysteine was continuously infused. The kidneys were removed and immediately snap-frozen. Tissue contents of AdoHcy, AdoMet, adenosine and adenine nucleotides were analyzed by means of HPLC. Under normoxic condition the tissue contents of AdoHcy, AdoMet and adenosine were 0.7±0.05, 44.1±1.0 and 3.8±0.1nmol/g wet weight, respectively. Renal ischemia for 30min resulted in an increase of AdoHcy levels from 0.7±0.05 to 9.1±0.6nmol/g wet weight and in a dramatic decrease of the AdoMet/AdoHcy ratio and energy charge from 65.1±5.6 to 2.8±0.2 and from 0.87±0.01 to 0.25±0.01, respectively. Application of exogenous adenosine into the ischemic kidney did not result in further AdoHcy accumulation. However, when homocysteine was infused into the ischemic kidney, AdoHcy increased five-fold above control levels, during 5min ischemia. Systemic infusion of homocysteine leads to a reduction of the methylation potential also in the normoxic kidney. We conclude that (i) the methylation potential in the kidney is markedly reduced during ischemia, mainly due to accumulation of AdoHcy; (ii) elevation of AdoHcy tissue content during ischemia is the result of the inhibition of AdoHcy hydrolysis; (iii) homocysteine is rate limiting for AdoHcy synthesis in the ischemic kidney; (iv) under normoxic conditions hyperhomocysteinemia can affect the methylation potential in the renal tissue.