Antiserum (A.TL x B10)F1 anti B10.HTT, obtained after skin grafting and immunization with spleen and lynph node cells, contains antibodies against antigens controlled by the I region of the H2 complex (Ia antigens). Two additional Ia antigens have been identified, antigen Ia.3 which is restricted to the H2(s) haplotype and its recombinant derivatives H2(t2) and H2(t3), all showing the same Ir 1A(s) subregion, and antigen 1a.4 which is shared by H2 haplotypes f, r, and u. The anti Ia antibodies react in a direct cytotoxic test with about 40 to 50% lymph node cells and 50 to 60% spleen cells, but do not react with thymus or bone marrow cells. However, the anti iA antibodies can be absorbed in vitro with large numbers of cells by both thymus and bone marrow cells; they cannot be absorbed with muscle, erythrocytes and brain tissue. After specific removal of anti Thy 1.1 sensitive cells from the spleen cell suspension, the proportion of cells killed by the anti Ia sera increases to almost 90%. Pretreatment in vivo with cyclophosphamide abolishes the anti Ia reactivity of spleen cells. Suspensions filtered through a nylon fiber column are enriched in Ia negative cells, the Ia positive cells being retained on the fibers. Pretreatment in vivo with hydrocortisone results in the appearance of a small fraction of Ia positive cells in the thymus. Thus, the Ia antigens are probably restricted to lymphocytes with predominant expression on B cells; if T cells express these antigens at all they do so either with a very low surface density or only in a minor subpopulation.