Recent studies in molecular genetics have revealed the striking fact that the previously known I-J subregion (or I-J gene) does not exist at the exactly prescribed position in the I-region of the murine major histocompatibility complex (MHC). How, then, can we comprehend the established serological and functional significance of I-J genes and I-J products? To answer this question we reexamined systematically the specificities and functional activities of monoclonal anti-I-J antibodies. A series of monoclonal anti-I-Jk antibodies were newly established by fusion of B10.A(3R) spleen cells immune to B10.A(5R) lymphoid cells with P3X63-Ag8-653. Their abilities to eliminate known functions of T cell subsets and to react with I-J+ T cell clones of defined functions were examined in an in vitro secondary antibody response and by fluorescence-activated cell sorter analysis. The monoclonal antibodies (mAb) were divided into three groups: (1) those reactive with suppressor inducer T cells (Tsi), Tsi hybridomas, and the antigen-specific T cell factor (TsF) derived from them; (2) those specific for suppressor effector T cells (Tse) and Tse clones; and (3) those reactive with some but not all helper T cells. The determinants detected by these three different groups of antibodies are apparently present on separate, nonoverlapping cell populations and functionally distinct clones. The above results indicate the multiplicity of I-J products expressed on different T cell subsets, and sharply contradict the notion, derived from molecular genetics, that not even a single gene can be accommodated in the I-J subregion. To resolve this dilemma, we compared the above results with those obtained with another set of mAb that also detected I-region-controlled determinants on augmenting and helper T cells. Although the specificities of these mAb clearly mapped to within the I-region, none of them reacted with conventional class II Ia antigens of B cells and macrophages. The common properties shared by these anti-Ik and anti-I-Jk antibodies are that (1) they react only with T cells and T cell clones with I-region-controlled functions; (2) they can block some of the Ia-restricted cell interactions, including those of helper and suppressor T cells; and (3) they inhibit the syngeneic and/or allogeneic mixed lymphocyte reaction (MLR) by blocking the responder but not the stimulator cells.(ABSTRACT TRUNCATED AT 400 WORDS)