Transplantation of positively selected, CD34(+) peripheral blood stem cells from alternative donors frequently results in delayed immune reconstitution. A shift towards a type 2 cytokine production might be a major contributing factor. We therefore decided to measure IFN-gamma, IL-2, IL-4, and IL-10 after stimulation of peripheral mononuclear cells with PMA/ionomycin and on a single cell level by intracellular cytokine staining during different stages of immune reconstitution. Immediately after transplantation, secretion of all selected cytokines was substantially diminished, and remained subnormal compared to controls until the end of the first year despite normalizing T-cell levels. IL-2 was predominantly produced by CD4(+)CD45RA(+) naïve, whereas IFN-gamma originated mainly from CD8(+)CD45RO(+) memory T cells. Secretion of IL-2 was correlated with the numbers of naive CD4(+) T cells, whereas IFN-gamma secretion correlated with total CD3(+) T-cell counts. IL-4 and IL-10 were produced by CD4(+) and CD8(+) memory T cells; secretion of these cytokines was low, however, and did not increase during follow-up. Therefore, a shift towards a preferential production of type 2 cytokines could not be observed. Analysis of CD69 upregulation upon stimulation revealed a deficiency in patient T-cell activation, which unexpectedly comprised both naïve and memory T-cell subpopulations. Therefore, we suggest that a defect in T-cell activation intrinsic to the host and not graft-versus-host disease, post-transplant immunosuppression or a shift towards a type 2 cytokine pattern contributes to the impaired production of cytokines post-transplant. Further studies will focus on the elimination of host factors that may adversely affect T-cell function after transplantation.