Heat shock protein (Hsp) 60 is thought to function as endogenous danger signal by activating professional antigen-presenting cells (APC) through toll-like receptor (TLR) 4 and CD14, a mechanism that is also used by bacterial LPS. We recently showed that Hsp60 binds LPS and enhances LPS-induced immune stimulation. On the other hand, we also observed immune stimulation by Hsp60 independent of LPS which was partially mediated by Hsp60-induced IFN alpha. Here, we study the mechanisms involved in immune stimulation mediated by endotoxin-free Hsp60. We show that T cell co-stimulation induced by LPS-free Hsp60 was independent of TLR4 and the TLR-associated myeloide differentiation factor 88-signaling pathway. LPS-free Hsp60 did not induce IL-6, IL-12 or tumor necrosis factor alpha production in APC nor were these cytokines needed for Hsp60-mediated T cell co-stimulation in the absence of LPS. In contrast to endotoxin-free Hsp60, T cell co-stimulation induced by LPS or Hsp60/LPS complexes strictly depended on IL-12 and functional TLR-4. Furthermore, we show that LPS-free Hsp60 enhances IFN alpha expression in APC and that this cytokine represents one important mediator in immune stimulation by Hsp60 in the absence of LPS. Taken together, we provide evidence that endotoxin-free Hsp60 and LPS or Hsp60/LPS complexes employ different signaling mechanisms to transduce co-stimulatory signals.