Soluble staphylococcal peptidoglycan (sPGN) is an inducer of cytokine secretion and may activate macrophages through the CD14 lipopolysaccharide (LPS) receptor. To elucidate sPGN-activated signal transduction pathways, stimulation of mitogen-activated protein (MAP) kinases by sPGN was studied in mouse RAW264.7 macrophages. sPGN strongly activated extracellular signal-regulated kinase (ERK) 1 and ERK2, moderately activated c-Jun NH2 terminal kinase (JNK), and weakly activated p38 MAP kinase, in contrast to LPS, which strongly activated all of these kinases, and phorbol 12,13-dibutyrate (PDB), which strongly activated ERK1 and ERK2 but did not activate p38 or JNK. sPGN- and LPS-induced activation of ERK1 and ERK2, unlike PDB-induced activation, was sensitive to inhibition by herbimycin A and insensitive to inhibition by increased intracellular cAMP. These results demonstrate differential activation of MAP kinases by sPGN, similar but not identical activation of signal transduction pathways by sPGN and LPS, and different mechanisms of MAP kinase activation by bacterial stimulants and phorbol esters.