The effect of Clostridium perfringens alpha-toxin on production of tumor necrosis factor (TNF)-alpha and nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells was studied. The pretreatment of wild type alpha-toxin, but not the inactive mutant, significantly decreased LPS-induced TNF-alpha and NO production. alpha-Toxin inhibited the expression of TNF-alpha and an inducible type of NO synthase protein and mRNA. Furthermore, it inhibited the phosphorylation of IkappaB-alpha and p65 NF-kappaB subunit, and the NF-kappaB luciferase reporter gene activity in LPS-stimulated cells. The pretreatment of alpha-toxin increased the level of intracellular ceramide. Taken together, Clostridium perfringens alpha-toxin pretreatment was suggested to inhibit LPS-induced TNF-alpha and NO production through the inhibition of NF-kappaB activation. The relationship between alpha-toxin-induced intracellular ceramide generation and the NF-kappaB inhibition is discussed.