The antitumor activity of 2'-deoxy-2'-methylidenecytidine (DMDC), an inhibitor of DNA synthesis, was examined and compared with that of 1-beta-D-arabinofuranosylcytosine (ara-C) against various murine tumors and human tumor xenografts. Against P388 murine leukemia, repeated treatments of DMDC were more effective than its single administration. Interestingly, DMDC was effective against colon 26 murine carcinoma, M5076 murine reticulum cell sarcoma, LX-1 human lung cancer xenograft, and SK-Mel-28 human melanoma xenograft, which are less sensitive or refractory to ara-C, while DMDC was not more potent against murine leukemias P388 and L1210 than ara-C. The in vitro cytotoxic effects of DMDC and ara-C against L1210 leukemia cells were prevented dose dependently by deoxycytidine, suggesting that DMDC, like ara-C, may require phosphorylation by deoxycytidine kinase for antitumor activity. DMDC was effective against human and murine experimental tumor models, especially nonleukemic tumors refractory to ara-C, suggesting that DMDC will be a promising agent for the treatment of cancer.