We have previously shown that tolerance can be induced against acute experimental autoimmune encephalomyelitis (EAE) in Lewis rats by bone marrow-derived dendritic cells (DC) that have been pulsed in vitro with encephalitogenic myelin basic protein peptide 68–86 (MBP 68–86), and injected subcutaneously into healthy rats prior to immunization with MBP 68–86 plus complete Freund's adjuvant. To elucidate better the properties of tolerogenic DC, we here compared plastic-adherent DC with floating, non-adherent DC, which were cultured for 7 days in the presence of granulocyte–macrophage colony-stimulating factor plus interleukin-4 (IL-4). Adherent DC expressed high levels of IL-10 mRNA and protein, and low levels of IL-12 mRNA and showed high expression of CD54 compared with floating DC. Proliferation, nitrite concentration and capacity for antigen presentation were lower in adherent DC than in floating DC. There were no differences between adherent and floating DC regarding expression of CD11c, OX62, major histocompatibility complex class II, CD80, or CD86. Most importantly, we observed that adherent DC induced tolerance to EAE in vivo when injected subcutaneously into Lewis rats prior to immunization, while floating DC did not. Adherent DC-mediated tolerance to EAE was associated with augmented proliferation, nitric oxide production and frequency of apoptotic cells as well as with up-regulation of transforming growth factor-β (TGF-β) -expressing cells in T-cell areas of lymph nodes. Tolerance induction by adherent DC seems to be related to a nitric oxide–apoptosis pathway and to up-regulation of TGF-β-expressing cells.