As the first step toward understanding the identity and functions of thymic macrophages in situ, we examined the phenotypic heterogeneity of mouse thymic macrophages in tissue sections by the immunohistochemical double staining method with four monoclonal antibodies (F4/80, Mac-2, anti-CD32/16 and anti-I-A antibodies) as macrophage markers. Morphologically, three types of macrophages were identified: dendritic, round and flat-shaped. Dendritic macrophages were scattered throughout the thymus, and most of them were stained by all four markers. Among these macrophages, those at the cortico-medullary region (CMR) expressed a high intensity of CD32/16 antigen. Round macrophages were also distributed throughout the thymus; most of them, however, were localized in the cortico-medullary region to the medulla. These cells were F4/80-negative, Mac-2-positive, CD32/16-negative and I-A-positive. In contrast, round macrophages located at the cortex expressed F4/80. Flat-shaped macrophages were localized at the subcapsular region of the cortex where active lymphopoiesis was observed. This type was positive for F4/80 and CD32/16, but negative for Mac-2. Furthermore, most of the three types of thymic macrophages showed intense reactions of the I-A antigen within the cytoplasm in addition to the expression of I-A antigen on the cell membrane. These results indicate that morphological characteristics of thymic macrophages at different locations reflect phenotypic variations detected in immunohistochemistry, and suggest that these different type macrophages may play distinct roles at various locations in thymocyte development in the thymus.