Antigen-presenting cells are of crucial importance for the initiation and regulation of regional immune responses. In a previous study, indirect morphological evidence that morphologically normal human orbital tissues contain HLA-DR-positive macrophages, which may represent antigen-presenting cells, has ben obtained. In the present study, these cells were characterized in detail using double immunoenzyme staining techniques with monoclonal antibodies directed against several well-characterized monocyte/macrophage markers and against HLA-DR gene products. The orbital muscular tissues appear to contain numerous HLA-DR, monocyte/macrophage marker double-stained cells, which are considered to be potential antigen-presenting cells. The cells are widely distributed in the connective tissue of all the orbital muscular tissues studied and consist of several subsets with different phenotypes. Furthermore, site-specific differences were shown between recti muscles and the levator/Müller's muscles with respect to the distribution of HLA-DR and one monocyte/macrophage marker (OKM5). Many of the orbital antigen-presenting cells appear to be of the dendritic type and are considered to be of major importance in regulating local orbital immunity.