Cellular methylation imbalance is associated with tumor progression, hepatic cancer, and cardiovascular disease. S-Adenosylhomocysteine (SAH) is an inhibitor of cellular methyltransferases, and increasing evidence suggests that SAH rather than homocysteine (Hcy) plays a crucial role in mediating these disorders related to methylation imbalance. The anti-metastatic gene nm23-H1 was recently identified in murine and human cancer lines, and the expressions of nm23-H1 mRNA and protein have been shown to be useful tumor invasion markers. We investigated the relationships of tumor cell invasion activities with the intracellular levels of SAH and Hcy and the level of DNA methylation (measured as the cellular content of 5-methyldeoxycytidine, 5-mdc) in four hepatocarcinoma cell lines (Sk-Hep1, J5, Hep-G2, Hep-3B) and one normal liver cell line (Chang's liver cells) with different invasion activities (Sk-Hep1 > J5 > Hep-G2 = Hep-3B > Chang's liver cells). We found that the intracellular level of SAH was the highest in SK-Hep1 cells and was correlated with the invasion activities (r = 0.75, P = 0.008), whereas the level of intracellular Hcy was the highest in Chang's liver cells and was not significantly correlated with the invasion activities of these cell lines (r = 0.24, P = 0.38). The levels of 5-mdc increased with decreasing invasion activities of these cell lines (r = 0.82, P = 0.002), that is, the order of DNA hypomethylation in these cell lines was Sk-Hep1 > J5 > Hep-G2 = Hep-3B > Chang's liver cells, because the lower levels of 5-mdc% represent the higher DNA hypomethylation. Thus, our results demonstrate that SAH rather than Hcy is associated with invasion activities of hepatoma cells, and they suggest that SAH may play an important role in the invasion activities through DNA hypomethylation.