In this work, we used colon carcinoma cell-line HCT116 to study the involvement of the 86-kDa subunit (Ku86) of DNA-protein kinase (DNA-PK) in human tumoural cell proliferation. We transfected these cells with a 639-bp cDNA encoding a Ku86 portion inserted into pcDNA3.1 vector in the antisense orientation. After selection by neomycin, we obtained more than 300 resistant colonies. In the Y'A5 colony that we chose as total population, we showed by PCR and RT-PCR that pcDNA3/Ku86 antisense was integrated in genomic DNA and that transcript was present. After cloning, we selected two clones, A20 and A23, which contained significatively reduced level of Ku86 protein. These two clones displayed a reduced DNA-PK activity from 44% to 71% and a slower growth than control cells. These results suggest that the HCT116 cell-line is a useful tool to investigate the role of Ku86 in the regulation of human tumoural cell growth.