Retroviral vectors encoding the herpes simplex thymidine kinase gene have been used to render T cells sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used in clinical trials for their graft-versus-leukaemia effects following allogeneic haematopoietic stem cell transplantation. In the event of graft-versus-host disease (GVHD) the cells were susceptible to elimination through exposure to ganciclovir. We have investigated the impact of T-cell activation, required for successful retrovirus-mediated gene transfer, on T-cell receptor repertoire profile, subset distribution and antiviral potential. Using a combination of antibodies against CD3 and CD28, T cells were transduced at high efficiency when exposed to retrovirus between 48 and 72 h later. Lymphocytes had undergone up to seven cycles of cell division by the end of the procedure. Although the T-cell receptor Vbeta repertoire was not altered after retroviral transduction, there were notable shifts in subset profiles with an increased proportion of CD45RO cells in transduced populations. T cells continued to proliferate for several days after transduction and were difficult to sustain under the extended culture conditions required to generate virus-specific T cells. These observations may explain the lower than expected levels of GVHD and poor antiviral immunity reported in recent trials.