The authors have previously shown the role of human immunodeficiency virus type 1 (HIV-1) Nef protein as a growth inhibitor to CD4+ T lymphocytes. We now report that the Nef antigen is partly expressed on both acutely and persistently infected human T cells. To investigate the cell surface expression of the Nef antigen, murine monoclonal antibodies (mAbs) were prepared by immunization with a recombinant Nef protein. The recombinant Nef was expressed by a baculovirus expression system as a truncated Nef protein with 22 kDa containing the middle to the C-terminus. Three clones were found to produce mAbs with IgM isotype against Nef protein by ELISA with the same truncated Nef. All these mAbs reacted on immunoblotting with two forms of Nef, 25 kDa and 27 kDa, in an HIV-1-infected human T-cell line and with the 27 kDa Nef in retroviral vector-infected mouse fibroblasts expressing a full-length Nef protein. Membrane immunofluorescence and flow cytometry with these mAbs revealed the Nef antigen to be expressed, at least in part, on the surface of these cells. Thus, the cell-surface form of Nef might play an important role in the selective depletion of CD4+ cells in HIV-1 infection.