CD40 is a 50 kDa transmembrane protein important for regulating B lymphocyte proliferation and differentiation. This novel activation antigen is primarily expressed by hematopoietic cells including B lymphocytes, follicular dendritic cells, and monocytes. Recently, human fibroblasts from a variety of tissues were shown to display CD40; however, its function was unknown. Cellular responses mediated by CD40 are naturally triggered by its counter-receptor, the CD40 ligand, which is displayed on activated T cells, mast cells, eosinophils, basophils, and B lineage cells. This study investigated the functional significance of CD40 expression on periodontal fibroblasts, in the context of periodontal inflammation. The experiments reported herein demonstrate constitutive CD40 expression on cultured periodontal ligament (PDL) and gingival fibroblasts. Interestingly, cells of gingival origin displayed up to 13-fold higher constitutive levels of CD40, versus fibroblasts from PDL. Interferon gamma (IFN gamma) treatment enhanced CD40 expression on PDL and gingival fibroblasts, with up to 61-fold induction of expression. Immunohistochemical staining was used to detect CD40 on fibroblastic cells in both normal and acutely inflamed gingival tissue. Expression of CD40 in inflamed tissue was significantly higher than in uninflamed tissue. Western blot analysis of anti-CD40 triggered cells revealed the induction of tyrosine phosphorylation on a 50 kDa protein in PDL and gingival fibroblasts. These results indicate that CD40 is an active signaling conduit in periodontal fibroblasts. This concept was further substantiated by the fact that CD40 engagement stimulated interleukin 6 (IL-6) production by gingival fibroblasts, but not periodontal ligament fibroblasts. Overall, these results demonstrate that CD40 on periodontal fibroblasts may functionally interact with CD40L-expressing cells. This CD40/CD40L interaction can stimulate fibroblast activation and synthesis of the proinflammatory cytokine IL-6.