APO-1/Fas (CD95) is a member of the tumor necrosis factor/nerve growth factor receptor superfamily and mediates apoptosis in various cell types. Here we show that L929 cells, expressing human APO-1 treated with agonistic antibodies (anti-APO-1), elicit an early and transient increase of S-adenosylhomocysteine (AdoHcy), a potent inhibitor of S-adenosylmethionine (AdoMet)-dependent methylation reactions. In contrast, anti-APO-1 did not induce an AdoHcy increase in L929-APO-1 Delta4 cells expressing a C-terminally truncated APO-1 lacking part of the 'death domain' known to be required for the transduction of apoptotic signals. Addition of adenosine and D, L-homocysteine also led to an increase of cellular AdoHcy thus enhancing anti-APO-1-induced killing of L929-APO-1 cells. Treatment with anti-APO-1 also induced release of arachidonic acid from phospholipids: this effect was augmented by elevated levels of AdoHcy. In contrast, AdoHcy had only a minor effect on anti-APO-1-mediated DNA fragmentation. These findings suggest that AdoHcy functions as a physiological modulator of APO-1-mediated cell death in L929 cells and enhances anti-APO-1-induced cell killing at least partially by acting via the phospholipase A2 pathway.