We examined whether the administration of methotrexate (MTX) prior to the injection of 5-[125I]iodo-2'-deoxyuridine (125IUdR) in rats with intrathecal (i.t.) TE-671 human rhabdomyosarcoma would enhance 125IUdR uptake by tumor cells and augment the therapeutic efficacy of this Auger-electron-emitting radiopharmaceutical. TE-671 cells were exposed in vitro to medium +/- MTX, and the percentage of cells in various phases of the cell cycle and the uptake of 125IUdR assessed. In addition, nude rats were injected i.t. with TE-671 cells and later infused i.t. with saline or MTX for 24 h prior to 125IUdR injection, and the radioactivity associated with their spinal cords was determined. Exposure of tumor cells in vitro to MTX leads to an increase in the uptake of 125IUdR as a consequence of both a rise in the absolute uptake per cell and an increase in the percentage of S-phase cells. A corresponding increase of radioactivity within the spinal cords of tumor-bearing rats also occurs in the presence of MTX. Tumor-bearing animals were infused/injected with MTX and/or 125IUdR, and the onset of paralysis was determined as a function of time. We find that: (i) MTX infusion leads to a slight increase in time to onset of paralysis (median [M] = 24 vs. 22 days, p = 0.79); (ii) 125IUdR injection results in a statistically significant delay (p < 0.01) in the onset of paralysis (M= 39 days); (iii) MTX administration prior to 122IUdR injection further increases the therapeutic efficacy (M = 45 days).