To examine the B-cell stimulatory properties of the regulatory Nef protein of HIV-1. The effect of the HIV-1 regulatory proteins Nef, Tat and Vif, were analyzed for their ability to induce differentiation of normal B lymphocytes into immunoglobulin secreting cells (ISC). A recombinant Nef protein, but neither Tat or Vif, was able to induce ISC in peripheral blood lymphocyte (PBL) cultures of HIV-1-seronegative donors. Another recombinant Nef protein, d-Nef, with a truncated amino terminal (deletion of 34 amino acids) failed to induce B-cell differentiation. Pretreatment of the Nef protein with a polyclonal anti-Nef-antibody abrogated its B-cell stimulatory activity. The Nef-induced B-cell differentiation was dependent on cell-to-cell contact. Cell surface molecules leukocyte function-associated molecule (LFA)-1, intracellular adhesion molecule (ICAM)-1, human lymphocyte antigen-DR and B7 were involved in the T-B-cell interaction because monoclonal antibodies to these molecules abrogated the Nef-induced B-cell differentiation response. The Nef protein was able to induce interleukin (IL)-6 messenger (m)RNA and IL-6 protein secretion in PBL, with monocytes as the primary source. These findings indicate that regulatory (Nef) proteins of HIV-1 contribute to the intense B-cell activation that occurs in association with HIV-1 infection. T-B-cell contact-dependent interaction and induction of IL-6 by these proteins appear to play major roles in this process.