Evaluation of selective killing of Herpes Simplex Virus 1 thymidine kinase (HSV1-tk) expressing tumors by radiolabeled 131I-fialuridine (FIAU), and of synergy between 131I-FIAU and Ganciclovir (GCV). HSV1-tk-expressing cell lines and parental cell lines were exposed to 131I-FIAU alone, GCV alone, or combinations. Activity and concentration were varied widely, concurrent and sequential administrations tested, and dose rate effects were studied. HSV1-tk-expressing cells accumulated up to 15.7-fold more 131I-FIAU, were growth inhibited by 2 μCi/ml, or 5 μCi/ml 131I-FIAU, and were inhibited by two log orders lower concentrations of GCV than parental cells. However, no synergy or additive effect was observed. Dose rate variations, or sequential treatment, did not alter outcome. Radioisotope therapy of HSV1-tk-expressing tumor cells with 131I-FIAU is reported for the first time. Lack of synergy between 131I-FIAU and GCV does not warrant further investigation of combination treatment with the two agents.