A series of B, T, natural killer (NK) cell, and monocyte-specific monoclonal antibodies was used to determine the localization of lymphocyte subpopulations in frozen tissue sections of human lymph node, spleen, tonsil, and thymus by means of an immunohistochemical technic. In thymus, most cortical thymocytes reacted with Leu 1, Leu 2a, Leu 3a, Leu 4, Lyt 3, OKT3, and OKT6 antibodies. Except for OKT6, Leu 2a, and Leu 3a, these antibodies also reacted with medullary thymocytes. The majority (70-80%) of medullary thymocytes reacted with Leu 3a and a smaller fraction (20-30%) with Leu 2a antibody. The staining pattern of thymic medulla approximates the staining pattern of peripheral T cells. In peripheral lymphoid tissues, the majority of cells in the paracortical region of lymph node and in the periarteriolar sheath of spleen stained with Leu 1, Leu 4, OKT3, and Lyt 3 antibodies. Staining with Leu 3a and Leu 2a identified 60-80% and 20-40% of total T cells, respectively, as defined by Lyt 3 positivity. In addition, a substantial number of Leu 3a+ and Leu 7+ cells were found in the germinal centers of secondary follicles. This finding supports the importance of these subsets of lymphocytes in regulation of the human immune response. Leu 2a+ cells were rare in tissues with prominent follicular hyperplasia, but appeared in considerable number in the red pulp of spleen. In the mantle zone of lymphoid follicles, the majority of lymphocytes were positive for IgM, IgD, and B1. Approximately 60-70% of these cells bore kappa chain and 30-40% lambda chain immunoglobulin. The extracellular substance in germinal centers was positive for B1, IgG, IgM, kappa, and lambda. The majority of germinal center cells appeared to contain no surface or cytoplasmic immunoglobulins. Small mononuclear cells bearing OKM1 marker were abundant in the marginal zone of white pulp and in the red pulp of spleen but, rarely were observed in other portions of lymphoid tissues. OKM1 also reacted with granulocytes. Leu 7+ (NK) cells were rare in the thymus, but frequent in the GC of secondary follicles. The distribution of Leu 7+ cells did not correspond to staining with Lyt 3 and Leu 2a.