Activation of mature T cells induces the expression of high affinity receptors for the T cell growth hormone, IL-2. A short term assay was used to determine the capacity of murine thymocyte subpopulations to express the gene encoding the 55-kDa chain of the IL-2R after stimulation in vitro. Thymocytes were cultured in the presence or absence of phorbol ester and calcium ionophore for 20 h, stained with antibodies against the IL-2R or the MHC Ag, H-2K, and analyzed by FACS. At least 90% of CD4-CD8- thymocytes were competent to express IL-2R, implying that this functional response is acquired early in development. However, at least 80% of cortical thymocytes were unable to express IL-2R before or after stimulation. Additional fractionation of the nonresponsive cells indicated that they constitute the great majority of the CD4+CD8+ population, including virtually all of the CD4+CD8+ proliferating blasts. Most nonresponsive cells remain viable and react to the chemical stimuli by increasing their surface expression of H-2K. The inability of most cortical thymocytes to express IL-2R suggests that a discrete loss of function occurs in most cells as they differentiate from CD4-CD8- precursors into the CD4+CD8+ class. The inferred loss of function is mediated at the level of IL-2 RNA accumulation and may be correlated with a pleiotropic alteration in physiologic response pathways.