Early events in the response of cells to lipopolysaccharide (LPS) include activation of NF-κB and stress-activated MAP kinase p38. Recent studies have shown that the human Toll-like receptor 2 (TLR2) mediates activation of NF-κB in response to commercial preparations of LPS (comLPS), membrane lipoproteins, and Gram-positive bacterial products. Here, we show that expression of TLR2 in human embryonic kidney 293 cells enabled p38 phosphorylation in response to comLPS, a synthetic bacterial lipoprotein, and B. subtilis. Activation of p38 was confirmed by an in vitro kinase assay using ATF2 as substrate and by an assay measuring activation of the downstream effector of p38, MAP kinase-activated protein kinase in cells. Thus, TLR2 initiated the signaling pathway for p38 in response to bacterial products.