Antiserum (A.TL × B10)F1 anti-B10.HTT, obtained after skin grafting and immunization with spleen and lymph node cells, contains antibodies against antigens controlled by the I region of the H-2 complex (Ia antigens). Two additional Ia antigens have been identified, antigen Ia.3 which is restricted to the H-2s haplotype and its recombinant derivatives H-2t2 and H-2t3, all showing the same Ir-1As subregion, and antigen Ia.4 which is shared by H-2 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.