Although proenkephalin A (PEA) messenger RNA (mRNA) has been detected in many types of immune cells, little understanding exists about its role or the role of enkephalin peptides in immune responses. We have studied the expression of PEA mRNA during thymocyte maturation by identifying the subpopulation of thymocytes that expresses PEA mRNA. PEA mRNA was induced in unfractionated murine thymocytes after in vitro activation of these cells with the T cell mitogen, concanavalin A (Con-A). A slight induction of PEA mRNA was seen after 48 h of Con-A stimulation; however, the maximal response occurred after 72 h of culture with Con-A. Two PEA mRNA bands were present in unfractionated thymocytes which had been cultured with Con-A for 48 and 72 h. The predominant band was 1.4 kilobases (kb), and a second band was approximately 1.7 kb. Fractionation of thymocytes into CD4, CD8, and double negative subpopulations showed that only the 1.4 kb PEA mRNA was inducible in the mature CD4 subpopulation. Induction required the presence of antigen-presenting cells in addition to CD4 thymocytes. Neither the 1.4 kb nor the 1.7 kb PEA mRNA was induced in the CD8 or double negative subpopulations. In contrast to the action of Con-A on murine thymocytes, PEA mRNA was not induced by this mitogen in murine splenic mononuclear cells at 24, 48, or 72 h. The regulated expression of PEA mRNA in murine thymocytes, but not in peripheral T lymphocytes, suggests a role for PEA mRNA and its peptides in thymocyte maturation.