We have previously shown that the infusion of donor lymphocytes expressing the herpes simplex virus thymidine kinase (HSV-tk) gene is an efficient tool for controlling graft-versus-host disease (GVHD) while preserving the graft-versus-leukemia (GVL) effect. In addition to the GVL effect, the administration of donor HSV-tk(+) cells could have a clinical impact in promoting immune reconstitution after T-cell-depleted stem cell transplantation (SCT). To explore this hypothesis, we have investigated whether in vitro polyclonal activation, retroviral transduction, immunoselection, and expansion affect the immune competence of donor T cells. We have observed that, after appropriate in vitro manipulation, T cells specific for antigens relevant in the context of SCT are preserved in terms of frequency, expression of T-cell receptor, proliferation, cytokine secretion, and lytic activity. A reduction in the frequency of allospecific T-cell precursors is observed after prolonged T-cell culture, suggesting that cell manipulation protocols involving a short culture time and high transduction efficiency are needed. Finally, the long-term persistence of HSV-tk(+) cells was observed in a patient treated in the GVL clinical trial, and a reversion of the phenotype of HSV-tk(+) cells from CD45RO(+) to CD45RA(+) was documented more than 2 years after the infusion. Based on all this evidence, we propose a clinical study of preemptive infusions of donor HSV-tk(+) T cells after SCT from haploidentical donors to provide early immune reconstitution against infection and potential immune protection against disease recurrence.