Antigens coded for by the major histocompatibility complex (MHC) are differentially expressed in the mouse thymus. Immunoperoxidase studies of frozen thymus sections incubated with monoclonal (hybridoma) anti-I-Ak antibodies revealed a dendritic straining pattern in the cortex and a confluent staining pattern in the medulla. Serial sections incubated with monoclonal anti-H-2Kk antibodies showed that H-2Kk antigens were only present at detectable levels in the medulla. Microenvironments expressing H-2Kk antigens also expressed I-Ak antigens. In cortico-medullary regions, relatively large MHC-negative areas were found. These areas appeared to connect to perivascular spaces surrounding blood vessels. Using a new postfixation labeling method for the detection of cell surface associated antigens on cells of the lymphoid system in situ, we have characterized the nature of MHC positive cell types at the ultrastructural level. These studies show that epithelial-reticular cells are the major MHC positive elements in the thymus. Lymphocytes in the medulla and in cortico-medullary bounderies are also MHC positive, however, lymphocytes in the cortex were not detectably labeled. These findings support the contention that epithelial-reticular cells are involved in the H2-restriction process during T cell maturation.