Stimulation of human immunodeficiency virus type 1 (HIV-1)-infected donor peripheral blood mononuclear cells (PBMCs) via the TCR-CD3 complex induces HIV-1 production in vitro (Zarling JM, et al.: Nature [London] 1990;347:92; Haffar OK, et al.: J Virol 1992;66:4279; Moran PM, et al.: AIDS Res Hum Retroviruses 1993;9:455). However, in addition to the primary stimulatory signal delivered through the TCR-CD3 complex, optimal T cell activation requires secondary or costimulatory signals delivered via various T cell accessory proteins (Alton A, et al.: Adv Immunol 1990;48:227). In this article we explore the role of costimulation of T cells via CD28 in HIV-1 replication. Ligation of CD28 with either a CD28-specific MAb or by coculture of PBMCs with Chinese hamster ovary (CHO) cell lines stably expressing either of the CD28 counterreceptors, B7-1 (CD80) or B7-2 (CD86), concomitant with stimulation via CD3, results in increased virus replication compared to stimulation via CD3 alone. CD28 ligation also augments de novo infection of CD3-stimulated seronegative donor PBMCs with cell-free virus. Increased virus replication following CD28 ligation is not solely attributed to increased levels of endogenous IL-2, because addition of an anti-IL-2-neutralizing antibody only partially inhibits the response. In contrast, interfering with the interaction between CD28 and its counterreceptors on antigen-presenting cells (APCs) using CTLA4Ig effectively inhibits virus replication. At high concentrations CTLA4Ig also reduces cell proliferation. These in vitro results suggest that CD28 plays a central role in HIV-1 replication and that interfering with the CD28 costimulatory pathway may modify the course of HIV-1 infection.