Using a complement-dependent cytotoxicity assay (CDC), we analyzed the presence of HLA-class II antigens on both stromal and hemopoietic cells in a miniaturized human long-term bone marrow culture system. 4-Hydroperoxycyclophosphamide (4-HC)-resistant hemopoietic stem cells capable of restoring in vitro hemopoiesis on irradiated stromal cell layers were HLA-DR, -DP, and -DQ negative. In addition, these cells failed to bind the monoclonal antibody (mAb) Tu 39, previously proposed as a candidate for the recognition of a novel class II antigen, '-DY'. On the other hand, the formation of confluent stromal cell layers was inhibited by HLA-DR- or -DP-specific mAbs, but not by the HLA-DQ-specific mAb Tu 22. This suggests the presence of HLA-DR- and/or HLA-DP-positive, but HLA-DQ-negative stromal precursor cells.