The interaction of CD28 with its ligands is important for T-cell activation. Recent studies demonstrated the existence of at least two ligands on accessory cells, CD80 (B7-1) and CD86 (B7-2). In this study we demonstrate that, although CD80 and CD86 are both expressed on monocytes, they seem to have different functions. Freshly isolated monocytes express CD86 but are CD80-negative. CD80 expression is weakly induced after 6–8 hr of in vitro culture and is enhanced by stimulation. CD86 expression is enhanced faster than CD80 expression and reaches the peak level after 4–6 hr in stimulated cells. Reverse transcription–polymerase chain reaction studies demonstrate that freshly isolated monocytes contain no CD80-mRNA. The mRNA of CD80 is induced after 4–6 hr of culture, which matches with the expression of the protein. Inhibition studies using different antibodies against both molecules and the fusion protein CTLA4Ig show that only anti-CD80 and CTLA4Ig could partially inhibit antigen-specific (tuberculin) and polyclonal (anti-CD3) lymphoproliferation and interferon-γ (IFN-γ) secretion of T cells cocultured with autologous monocytes. IFN-γ secretion was more sensitive to blocking costimulation than proliferation. The antibody BB-1 did not inhibit proliferation and cytokine secretion, nor did the anti-CD86 clone IT2.2. CTLA4Ig, which binds both CD80 and CD86, has the same inhibitory capacity as the anti-CD80 antibody tested. From those findings we conclude that human monocytes use CD80 as a costimulatory ligand for CD28 and utilize other costimulatory mechanisms besides those mediated via molecules of the B7 family.