Impaired T-cell function after T-cell– depleting (TCD) therapy has been hypothesized to be related to a transient predominance of extrathymically expanding memory T cells. To test whether after TCD therapy the naive T-helper cell population is functionally intact, the in vitro immune response of CD4⁺CD45RA⁺ (naive) and of CD4⁺CD45RA⁻ (memory) cells to polyclonal mitogens (immobilized anti-CD3, phytohemagglutinin) was analyzed by flow cytometry in 22 pediatric patients after high-dose chemotherapy (including 5 after autologous and 5 after allogeneic stem cell support). At 1 to 3 months after TCD therapy, patient samples showing decreased lymphoproliferative responses also showed a reduced induction of the early activation marker CD69 by CD4⁺ T cells from 4 to 72 hours after stimulation even when supplemented with exogenous interleukin-2. This defect affected CD4⁺CD45RA⁻ cells, but, strikingly, also CD4⁺CD45RA⁺ cells, including samples in which CD4⁺CD45RA⁺ cells were more than 90/μL, thus indicating ongoing thymopoiesis. Histogram analyses showed the median peak channel of CD69 in control CD4⁺CD45RA⁺cells rising 98-fold (median) but only 28-fold in patient cells (P < .0001). Apoptosis as detected by annexin V staining was increased in resting patient CD4⁺ T cells (25% versus 6%) and also affected CD4⁺CD45RA⁺ cells (12% versus 5%, P < .01). When peripheral blood mononuclear cells (PBMCs) were enriched for T cells, stimulatory responses of CD4⁺ cells and of CD4⁺CD45RA⁺ cells markedly improved. Thus, after TCD therapy suppressor factors contained in the non–T-cell fraction of PBMCs may affect T-helper cells irrespective of their naive or memory phenotype thus extending T-cell dysfunction to the presumably thymus-dependently regenerated T cells.