We demonstrated that a 65-kDa cytosolic protein (pp65) was most heavily phosphorylated in murine macrophages in response to bacterial lipopolysaccharide (J. Immunol. 146:3617, 1991). The pp65 phosphorylation closely correlated with cellular responses in the macrophages. We have thus performed amino acid sequence analysis of the two pp65-derived peptide fragments in order to elucidate its structure and function. The results indicated that pp65 is a novel protein existing almost exclusively in hemopoitetic cells and has the strong homology with human L-plastin, a substrate which has recently been identified as a novel protein transformation-dependently expressed in neoplastic human cells. Our pp65 is apparently the first purified protein whose phosphorylation has been augmented by stimulation with bacterial lipopolysaccharide and whose primary structure has been clarified. Because L-plastin possesses the unique functional domains, detailed investigation of the pp65 phosphorylation should lead to progress in our understanding of the mechanisms of macrophage activation.