Forssman (Fo) glycolipid antigen, as detected by a monoclonal antibody (mAb), is expressed by a subpopulation of murine macrophages in the spleen and peripheral lymph nodes. The histological distribution of Fo antigen in spleen and lymph nodes was studied by immunostaining of cryosections, and was compared with the staining pattern of four other mAbs known to recognize macrophage subpopulations: F4/80, Mac-1, MOMA-1, and ERTR-9. Fo+ macrophages were found exclusively in the red pulp of the spleen and the medulla of inguinal and axial lymph nodes. Macrophages in the other lymphoid organs were Fo-. Besides macrophages, reticular cells in T-dependent areas of spleen and lymph nodes were Fo+. Attempts to grow colonies of Fo+ macrophages from either bone marrow or spleen precursors were negative. While the usual number of F4/80+ colonies was obtained, only a few, small clusters of Fo+ macrophages were formed, which speaks against an early commitment of precursors to express Fo.