We previously demonstrated that Gram-positive bacteria activated immune cells via CD14 and Toll-like receptor 2 (TLR2). Although peptidoglycan, a major constituent of the bacterial cell wall, substituted for whole organisms, the essential structure of muramylpeptides required to stimulate the cells is not clear. We further investigated the critical determinant for recognition by CD14 and TLR2. Chinese hamster ovary (CHO) fibroblasts, which do not express a functional TLR2 transcript, were transfected with TLR2 or TLR4. These cells were exposed to freeze-dried Staphylococcus epidermidis and were subsequently subjected to the pro-inflammatory transcription factor nuclear factor-kappaB (NF-kappaB)-dependent CD25 expression assay. Heterologous expression of human TLR2, but not TLR4, in CHO cells conferred immune responsiveness to freeze-dried S. epidermidis. A preparation of peptidoglycan from S. epidermidis substituted for whole organisms. Staphylococcus aureus lytic enzyme-digested product (SEPS) from peptidoglycan retained the activity, but hydrolysis of the glycan backbone in SEPS by M-1 endo-N-acetylmuramidase resulted in loss of the activity. These findings showed that cellular activation by Gram-positive cell wall components was mediated by TLR2, but not TLR4, and indicated that the glycan backbone of peptidoglycan is critical for TLR2-mediated NF-kappaB activation.