Soluble CD14 (sCD14) in serum is known to sensitize host cells to LPS. In the present study, the contributions of sCD14 and LPS-binding protein to a lipid A moiety from LPS preparations of periodontopathogenic Fusobacterium nucleatum sp. nucleatum were compared with that of Escherichia coli-type synthetic lipid A (compound 506). F. nucleatum lipid A was identified to be a hexa-acylated fatty acid composed of tetradecanoate (C-14) and hexadecanoate (C-16), similar to dodecanoate (C-12) and C-14 in compound 506. The two lipid A specimens exhibited nearly the same reactivity in Limulus amoebocyte lysate assays, though F. nucleatum lipid A showed a weaker lethal toxicity. Both lipid A specimens showed nearly the same activities toward host cells in the absence of FBS, though compound 506 exhibited much stronger activity in the presence of FBS, sCD14, or sCD14 together with LPS-binding protein. Furthermore, native PAGE/Western immunoblot assays demonstrated that F. nucleatum lipid A had a weaker binding to sCD14 as compared with compound 506. These results suggest that sCD14 is able to discriminate the slight structural differences between these lipid As, which causes their distinct host cell activation activities.