A panel monoclonal antibodies (mAbs) raised to rat thymic non-lymphoid cells has been shown to discriminate between distinct subpopulations of macrophages depending on their anatomic localization in the thymus. These reagents were used in this study to examine the expression of macrophage-associated antigens in primary culture of rat thymic stromal cells. The phenotype of both adherent macrophage (AM) monolayers and non-adherent cells (NAC) released in culture medium was studied at different time points after cultivation. More than 95% AM expressed ED1 and R-MC 38 antigens (pan-macrophage markers), class I MHC antigens (OX-18) and iC3b receptor recognized by OX-42 mAb. Most of them (70-85%) were reactive with ED2, R-MC 40, 41 and 42 mAbs specific for cortical and cortico-medullary zone (CMZ) macrophages. A much smaller percentage was positive with R-MC 43/44 and R-MC 46/47 mAbs staining CMZ/medullary macrophages and a subset of cortical macrophages, respectively. A minor subset of AM expressed class II MHC molecules which progressively decreased during cultivation. NAC were phenotypically heterogeneous. In comparison with adherent cells they contained a lower percentage of cortical/CMZ phenotype macrophages. In addition, NAC were slightly enriched in R-MC 43+ cells and more significantly expressed IA/E antigens (85-95%). ED3, R-MC 39 and 45 mAbs reactive with thymic macrophages in situ were mostly non-reactive with AM and NAC in culture.