Doxorubicin was encapsulated into liposomes composed of hexadecylphosphocholine:egg yolk phosphatidylcholine:stearylamine (HePC.EPC:SA) 10:10.0.1 (molar ratio) (1) and EPC:SA 10:0.1 (molar ratio) (2). Liposomal formulations 1 and 2, as well as free doxorubicin and free HePC, were tested in vitro against HCT116 human colon cancer cell lines and peripheral blood mononuclear cells (PBMCs) obtained from healthy donors, using the sulphorodamine B assay. The activity of doxorubicin was retained or slightly improved when entrapped into liposomes 1 and 2, while liposomal formulation 1 incorporating doxorubicin was found to be less toxic against normal cells. The liposomes were tested in vivo against human colon cancer xenografts in scid mice. The antitumor activities of liposomes 1 and 2 were statistically similar to that of free doxorubicin, but their toxicity was significantly lower. Based on these results, the combination of HePC and doxorubicin in one liposomal formulation may be justified for further evaluation.