Carbon tetrachloride (CCl(4))-induced acute hepatitis is assumed to involve two phases. The initial phase, initiated within 2 h after CCl(4) administration, involves the generation of reactive oxygen species. The second phase is assumed to start about 8 h subsequent to CCl(4) administration and involves the oxidant-induced activation of Kupffer cells, which release various pro-inflammatory mediators such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). We investigated the role of Kupffer cells during CCl(4) intoxication using Nucling-knockout mice (the KO group), in which the number of Kupffer cells is largely reduced. Plasma alanine transaminase and aspartate transaminase levels demonstrated that the liver necrosis during the second phase was significantly alleviated in the KO group compared with that in the wild-type mice (the WT group). Plasma TNF-α concentrations in the WT group significantly increased 24 h after CCl(4) intoxication, whereas those in the KO group did not significantly increase. Plasma IL-6 levels also significantly increased in the WT group 24 h after CCl(4) administration, but those in the KO group did not increase at any time point. These results indicated that excess reactions of Kupffer cells, once primed by oxidants, were involved in the exacerbation of oxidative stress and liver damage during the second phase of CCl(4) intoxication.