During T-cell maturation, thymocytes interact with thymic stromal major histocompatibility complex (MHC) determinants and thymic hormones, and proliferate, apparently in response to MHC gene products, in the absence of antigen. The maturing thymocytes also express a series of cell surface molecules, at one stage coexpressing T4, T6, and T8. Mature T cells express either T4 or T8, lack T6, bear the T3-Ti receptor complex on the cell surface, and require antigen in addition to MHC determinants to proliferate. To study whether DNA methylation may be involved in regulating phenotypic and functional changes observed during thymocyte maturation, cloned, T4+ Interleukin-2 dependent, antigen-specific T cells were treated with an inhibitor of DNA methylation, 5-azacytidine (5-azaC). The 5-azaC treated cells lost the requirement for antigen and could be activated by autologous macrophages alone. Anti-class II and anti-T3, but not anti-class I monoclonal antibodies, inhibited activation of 5-azaC treated T4+ cells by macrophages, implying that the T3-Ti receptor complex may be recognizing class II MHC molecules without antigen. No changes in T3 and T4 expression were noted, and neither T8 nor T6 was induced.