Various heterodinucleoside phosphates of 5-fluorodeoxyuridine (5-FdUrd) and arabinofuranosylcytosine (Ara-C) have recently been synthesized as potent chemotherapeutic agents. 5-Fluorodeoxyuridine is being used in patients with colorectal carcinoma, whereas Ara-C is one of the most effective agents in the treatment of hematological malignancies. We now investigated the action of three novel amphiphilic dimers with different structures in various 5-fluorouracil (5-FU) sensitive and resistant human colon tumor cell lines (CCL228, CCL227, 5-FU resistant CCL227 and HT-29) as well as in L1210 murine leukemia cells. Activity of the heterodimers was determined by clonogenic and growth inhibition assays including the induction of programmed cell death. In addition, the in vivo effects were tested in L1210 leukemia bearing mice. We show that these compounds inhibited the number of colonies of 5-FU sensitive and resistant human colon tumor cell lines with IC50 values ranging from 0.65 to 1 nM. The investigated dimers induced dose-dependent apoptosis in HT-29 colon tumor cells as well as in L1210 leukemia cells. No significant difference in the cytotoxicity of these agents could be observed between 5-FU sensitive and resistant cells, indicating that these compounds might be used in the treatment of 5-FU resistant tumors. In L1210 leukemia bearing mice the survival of tumor-bearing animals was significantly increased in comparison with untreated control animals. We therefore conclude that these new heterodinucleoside phosphates of 5-FdUrd and Ara-C might be an additional option for the treatment of sensitive and 5-FU resistant colon cancer and hematological malignancies.