AbstractToll‐like receptor 2 (TLR2) is a pattern recognition receptor that plays an important role in enabling cells of the innate immune system to recognize conserved structural motifs on a wide array of pathogens including gram‐positive bacteria. Although microglia have recently been shown to express TLR2, the functional significance of this receptor in mediating microglial activation remains unknown. To ascertain the importance of TLR2 in microglial responses to S. aureus and its cell wall product peptidoglycan (PGN), we evaluated primary microglia from TLR2 knockout (KO) and wild‐type (WT) mice. TLR2 was found to play a pivotal role in PGN recognition and subsequent activation in primary microglia, as demonstrated by the attenuated expression of TNF‐α, IL‐12 p40, MIP‐2, and MCP‐1 in PGN‐treated TLR2 KO microglia compared with WT cells. In contrast, the responses of TLR2 KO and WT microglia to S. aureus were qualitatively similar, indicating that alternative receptors are responsible for recognizing intact bacteria. Microarray analysis confirmed that TLR2 plays a central role in PGN recognition by primary microglia. The expression of MyD88, a central adapter molecule in TLR‐dependent signaling, was similar in both TLR2 KO and WT microglia, suggesting that the defect in PGN recognition by the former is not due to alterations in this key signaling intermediate. These findings reveal the complex nature of gram‐positive bacterial recognition by microglia, which occurs, in part, through engagement of TLR2. © 2004 Wiley‐Liss, Inc.