A central question in T cell development is what makes cortical thymocytes respond to stimulation in a qualitatively different way than any other thymocyte subset. Part of the answer is that AP-1 function changes drastically at two stages of T cell development. It undergoes striking down-regulation as thymocytes differentiate from immature, CD4(-)CD8(-) double-negative (DN) TCR- thymocytes to CD4(+)CD8(+) double-positive (DP) TCRlo cortical cells, and then returns in the cells that mature to TCRhigh, CD4(+)CD8(-) or CD4(-)CD8(+) single-positive (SP) thymocytes. At all three stages, the jun family mRNAs can be induced similarly. However, we demonstrate that DP cortical thymocytes are specifically impaired in c-fos and fosB mRNA induction, even when stimuli are used that optimize survival of the cells and a form of in vitro maturation. fra-2 expression is induction independent but much lower in DP cells than in the other subsets. Overall Fos family protein induction accordingly is severely decreased in DP cells. Defective c-Fos and FosB expression in cortical thymocytes is functionally significant, because antibody supershift experiments show that in activated immature and mature thymocytes, most detectable AP-1 DNA-binding complexes do contain c-Fos or FosB. Thus, defective c-Fos and FosB expression in cortical thymocytes qualitatively alters any AP-1 complexes they might express. The cortical thymocytes are not deficient in mRNA expression for any of the constitutive transcription factors that are known to be needed to drive c-Fos or FosB expression, so it is possible that the activity of these factors is developmentally regulated through a post-transcriptional mechanism.