Heat shock protein (HSP) 60 nonspecifically activates cells of the innate immune system. In the present study, we characterized the effects of human HSP60 maturation, cytokine release, and T cell-activating capacity of bone marrow-derived dendritic cells (DC). Furthermore, we analyzed HSP60-induced signal transduction in DC. HSP60 strongly stimulated DC for maturation and release of TNF-alpha, IL-12, and IL-1 beta. However, HSP60 elicited only a weak IL-10 response in DC suggesting a Th1 bias. HSP60-treated DC induced proliferation of allogeneic T cells. Again, a Th1 bias was noted in that cocultures of allogeneic T cells and HSP60-treated DC released IFN-gamma but only small amounts of IL-10 and no detectable IL-4. Signaling via Toll-like receptor 4 was involved in HSP60-induced cytokine release and maturation because DC of C3H/HeJ mice with a mutant Toll-like receptor 4 showed deficient response to HSP60. HSP60 was found to rapidly activate the mitogen-activated protein kinases p38, c-Jun N-terminal kinase, and extracellular signal-regulated kinase as well as I kappa B in DC. Phosphorylation of these signaling molecules was also mediated by LPS, but with much slower kinetics. Thus, HSP60 stimulates DC more rapidly than LPS and elicits a Th1-promoting phenotype. These results suggest that DC play a pivotal role in priming for destructive Th1-type responses at sites of local HSP60 release.