Previously, we have reported an antitumor efficacy of liposomal N-(phosphon-acetyl)-L-aspartic acid (or PALA) in C-26 tumor bearing Balb/c mice, where PALA in liposome was administered one day after tumor inoculation. In this report, we have investigated the therapeutic potency of liposomal formulation of PALA, which was administered eight days after tumor inoculation in the same C-26 tumor bearing mice. The C-26 murine colon tumor inoculated mice were randomized for the in vivo therapy and the survival was measured after a single intraperitoneal injection of the drug. When the therapy was initiated eight days after tumor inoculation, DSPC-PALA at 150 mg/kg resulted in a significant increase in median survival time (MST) of 56% over the control group which received MES/HEPES buffer alone. However, none of the free PALA and DSPG-PALA liposome doses caused a statistically significant increase in MST over control group at the 95% confidence level. At 750 mg/kg dose, free PALA caused a marginally significant improvement in MST by 34%, but both 375 mg/kg and 150 mg/kg doses of free PALA caused only a 2% and a 4% increase in MST, respectively. These results show that PALA in neutrally charged liposome can exhibit considerably greater potency than free PALA in established C-26 tumor bearing mice.