Although concanavalin A (Con‐A)‐induced experimental hepatitis is thought to be induced by activated T cells, natural killer T (NKT) cells, and cytokines, precise mechanisms are still unknown. In the current study, we investigated the roles of Kupffer cells, NKT cells, FasL, tumor necrosis factor (TNF), and superoxide in Con‐A hepatitis in C57BL/6 mice. Removal of Kupffer cells using gadolinium chloride (GdCl₃) from the liver completely inhibited Con‐A hepatitis, whereas increased serum TNF and IFN‐γ levels were not inhibited at all. Unexpectedly, anti‐FasL antibody pretreatment did not inhibit Con‐A hepatitis, whereas it inhibited hepatic injury induced by a synthetic ligand of NKT cells, α‐galactosylceramide. Furthermore, GdCl₃ pretreatment changed neither the activation‐induced down‐regulation of NK1.1 antigens as well as T cell receptors of NKT cells nor the increased expression of the CD69 activation antigen of hepatic T cells. CD68⁺ Kupffer cells greatly increased in proportion in the early phase after Con‐A injection; this increase was abrogated by GdCl₃ pretreatment. Anti‐TNF antibody (Ab) pretreatment did not inhibit the increase of Kupffer cells, but it effectively suppressed superoxide/reactive oxygen production from Kupffer cells and the resulting hepatic injury. Conversely, depletion of NKT cells in mice by NK1.1 Ab pretreatment did suppress both the increase of CD68⁺ Kupffer cells and Con‐A hepatitis. Consistently, the diminution of oxygen radicals produced by Kupffer cells by use of free radical scavengers greatly inhibited Con‐A hepatitis without suppressing cytokine production. However, adoptive transfer experiments also indicate that a close interaction/cooperation of Kupffer cells with NKT cells is essential for Con‐A hepatitis. Conclusion: Superoxide produced by Kupffer cells may be the essential effector in Con‐A hepatitis, and TNF and NKT cells support their activation and superoxide production. (HEPATOLOGY 2008;48:1979‐1988.)