We showed previously that dendritic cells (DCs) treated with mitomycin C (MMC) tolerize allogeneic T cells in vitro and this might be mediated by downregulation of CD80, CD86, and ICAM-1. Here we analyze the suppression of the T-cell response induced by MMC-DCs in vivo. Rats injected with allogeneic DCs developed a strong lymph node reaction, whereas MMC-DCs induced no reaction. The same effect was obtained when CD80, CD86, and ICAM-1 expressed by DCs were blocked with antibodies. One injection of donor MMC-DCs strongly prolonged heart allograft survival in a donor-specific manner. Suppression of rejection was also achieved when donor DCs were pretreated with a combination of anti-CD80, anti-CD86, and anti-ICAM-1 antibodies, showing that downregulation of these molecules confers the DCs inhibitory properties. We conclude that allogeneic MMC-DCs specifically inhibit the T-cell response in vivo and that downregulation of CD80, CD86, and ICAM-1 is a potential mechanism of this effect.