ONO-4007 is a new synthetic lipid A derivative with low endotoxic activities. We have reported that ONO-4007 could be a new bio-logical response modifier for the treatment of tumor necrosis factor (TNF)-&agr;-sensitive tumors. In this study, we confirmed that ONO-4007 activated a murine macrophage cell line, RAW264.7, and that the activated RAW264.7 cells produced TNF-&agr;in vitro. RAW264.7 cells stimulated for less than 15 min with ONO-4007 (40 μg/ml) did not produce TNF-&agr;(less than 4 U/ml). However, 24 h stimulation with ONO-4007 induced TNF-&agr;production (more than 256 U/ml) in RAW264.7 cells. Although P38 in mitogen-activated protein kinase of the RAW264.7 cells was not tyrosine phosphorylated by ONO-4007 stimulation, ERK1 of the RAW264.7 cells was tyrosine phosphorylated for 5–15 min by ONO-4007 stimulation. Tyrosine phosphorylation of ERK1 decreased gradually from 15 min after stimulation and almost disappeared 60 min after stimulation. These findings indicate that ONO-4007 stimulates RAW264.7 cells immediately and induces tyrosine phosphorylation of ERK1 in the RAW264.7 cells for 5–15 min. These data suggest that the signal transduction pathway of ONO-4007 may be similar to that of lipopolysaccharide.