Functionally mature T cells respond to stimulation via the Ag receptor by secretion of IL-2 and/or other lymphokines and by proliferation. However, immature CD4+8+ thymocytes do not secrete IL-2 or proliferate in response to stimulation. We have analyzed murine thymocyte populations enriched for CD4+ and CD4+8+ cells as well as the functionally mature CD4+ lymphoma C6VL-B and the CD4+8+ lymphoma 1010 for their ability to express mRNA related to early products of T cell activation signals. When stimulated with the calcium ionophore (Ionomycin) plus PMA, all the cells, regardless of their phenotype, accumulated abundant levels of c-myc mRNA. However, in contrast to the CD4+ thymocytes and C6VL-B, which accumulated abundant levels of IL-2 transcripts, neither the normal CD4+8+ thymocytes nor 1010 expressed IL-2 mRNA before or after stimulation. We have also examined these cells for the presence of the murine equivalents of two nuclear DNA-binding factors, NFAT-1 and NFIL2-A, which have been shown to be involved in IL-2 gene expression in human T cells. Our results indicate: 1) NFIL-2A binding activity is constitutively expressed in both CD4+ and CD4+8+ thymocytes and lymphomas and 2) NFAT-1 binding activity is readily detected in CD4+ thymocytes and C6VL-B, but is detected in very minimal amounts in populations enriched for CD4+8+ thymocytes and in 1010 upon activation. These results suggest that the failure of CD4+8+ thymocytes to express IL-2 mRNA upon stimulation may be in part due to the lack of inducibility of NFAT-1 binding activity, and that functional maturation of this population might be associated with acquisition of the ability to induce NFAT-1 activity.