Chlamydia pneumoniae stimulates potently maturation of and cytokine secretion by bone marrow-derived dendritic cells (BMDDC). BMDDC responses depend mainly on Toll-like receptor (TLR)2 and to a minor extent on TLR4. We demonstrate here using C. pneumoniae in an infectious model with the replication-permissive epithelial cell line HEp2 that HSP60 is produced in substantial amounts in chlamydial inclusions during infection. Electron microscopy of chlamydial inclusions revealed that HSP60 was mainly associated with reticulate bodies, but was also located in between the different chlamydial developmental forms. Supernatants of permissive HEp2 cells infected with C. pneumoniae contained soluble chlamydial HSP60 as demonstrated by Western blotting and were able to stimulate BMDDC of wild-type mice. The stimulatory capacity of culture supernatants correlated with the presence of chlamydial HSP60. In contrast, BMDDC from TLR4-mutant mice crossed to TLR2-deficient mice were not stimulated by the culture supernatant, indicating that chlamydial HSP60 but not cytokines, possibly secreted by infected HEp2 cells, are responsible for the observed stimulation of BMDDC. Purified recombinant HSP60 from C. pneumoniae stimulated BMDDC in a TLR2- and TLR4-dependent fashion similar to the whole microorganism. In summary, these data suggest chlamydial HSP60 as an important mediator of inflammatory responses during infection with C. pneumoniae.