This is the first report on gene therapy of mice melanoma by HSV - tk/ACV system. The sensitivity to ACV of genetically modified B16 cells (B16LNTK) was much higher than that of the parental cells. The sensitivity to ACV of B16 cells was increased when they were co - cultured with B16LNTK cells with various ratios. which showed the exist of the by stander effects. The tumor volume of B16LNTK (0.25cm3) is 94% less than that of B16 in C57BL/6 mice after 20-days ACV treatment. (P< 0.01) . B16 tumor loaded mice were treated in situ with HSV-tk /ACV, a significant effect was obtained. The tumor volume of B16 decreased to 57% of its origin. (P < 0. 05) The above results showed the HSV - tk/ACV system is a promising method for gene therapy of mice melanoma.