Homocysteine (Hcy), S-adenosylhomocysteine (SAH) and adenosine (Ado) are methionine metabolism intermediates that may act synergistically in certain disease. In this study, we examined whether HCy, SAH and Ado may synergistically induce neuronal apoptosis of BV-2 microglial cells. We found that an incubation of BV-2 cells with 1 mM Hcy, 1 muM SAH and 100 muM Ado (SAH + Hcy + Ado) led to marked apoptosis of BV-2 cells, as evidenced by several markers of apoptosis. A synergistic effect of SAH + Hcy + Ado on apoptosis (2.55-fold, P < 0.05) was obtained, as calculated using the data of Annexin V-positive cells. This combination markedly induced intracellular levels of reactive oxygen species (ROS) starting at 6 h and significantly decreased the mitochondrial potential starting at 12 h. The combination significantly elevated caspase-9 and caspase-3 activities at 24 and 48 h. The combination also induced hypomethylation (at 24 and 48 h), as indicated by significantly decreased 5-methyldeoxycytidine levels and SAM/SAH ratios. Pre-incubation of cells with alpha-tocopherol (30 muM) reduced the increase of ROS (at 6 h) and significantly restored cell viability (at 24 and 48~h) in the SAH + Hcy + Ado group. Overall, the present study demonstrates that SAH, Hcy and Ado synergistically induce BV-2 apoptosis, possibly by generation of ROS and induction of intracellular hypomethylation.