The steroidogenic enzyme 21-hydroxylase (21OH) is the main autoantigen in autoimmune primary adrenal failure (Addison's disease). Autoantibodies against 21OH are immunological markers of an ongoing autoimmune process but are not directly involved in the tissue destruction. Autoreactive T cells are thought to mediate tissue damage, but the T cell antigen(s) has not been identified. To find out whether 21OH contains important immunodominant epitopes for T cells, we first immunized BALB/c and SJL inbred mouse strains with recombinant 21OH and showed that lymph node cells proliferated effectively following in vitro stimulation with recombinant 21OH (stimulation indices (SI) 20-40). We further synthesized a series of peptides based on 21OH with amino acid sequences with propensity to bind to major histocompatibility complex class II molecules. Only a few peptides could trigger lymphocytes of 21OH-primed mice to proliferate. One of these, 21OH (342-361), stimulated effectively 21OH-primed lymph node cells of SJL mice (SI = 4-8) and also, although to a lesser extent, of BALB/c mice (SI = 2.5). When SJL mice were immunized with 21OH (342-361), the immunizing peptide as well as peptide 21OH (346-361) triggered a significant proliferative response (SI = 24). A peptide from another part of 21OH, namely 21OH (191-202), did not stimulate the 21OH (342-361)-primed cells. Moreover, stimulation of lymph node cells of mice immunized with 21OH (342-361) with 21OH resulted in a significant proliferative response. We conclude that 21OH (342-361) is an immunodominant determinant for T cells in SJL and probably BALB/c mice. 21OH (342-361) corresponds to the substrate binding site of the enzyme. The p342-361 region may be involved in the pathogenesis of autoimmune adrenal failure in humans.