We have investigated the ability of hyaluronic acid (HA) fragments to activate the transcription factor NF-κB. HA fragments activated NF-κB in the cell lines T-24, HeLa, MCF7, and J774. Further studies in T-24 cells demonstrated that HA fragments also induced IκBα phosphorylation and degradation, κB-linked reporter gene expression, and ICAM-1 promoter activity in an NF-κB-dependent manner. The effect of HA was size dependent as neither disaccharide nor native HA were active. CD44, the principal cellular receptor for HA, was critical for the response because the anti-CD44 Ab IM7. 8.1 blocked the effect on NF-κB. HA fragments activated the IκB kinase complex, and the effect on a κB-linked reporter gene was blocked in T-24 cells expressing dominant negative IκB kinases 1 or 2. Activation of protein kinase C (PKC) was required because calphostin C inhibited NF-κB activation and IκBα phosphorylation. In particular, PKCζ was required because transfection of cells with dominant negative PKCζ blocked the effect of HA fragments on κB-linked gene expression and HA fragments increased PKCζ activity. Furthermore, damnacanthal and manumycin A, two mechanistically distinct inhibitors of Ras, blocked NF-κB activation. Transfection of T-24 cells with dominant negative Ras (RasN17) blocked HA fragment-induced κB-linked reporter gene expression, and HA fragments activated Ras activity within 5 min. Taken together, these studies establish a novel signal transduction cascade eminating from CD44 to Ras, PKCζ, and IκB kinase 1 and 2.