It has recently been suggested that heat shock protein (Hsp) 70, an intracellular protein, can be released into the extracellular compartment and exert important immunomodulatory functions. Although elevated Hsp70 has been found in synovial fluid from patients with rheumatoid arthritis (RA), its sources and extracellular functions remain unclear. In this study, we explored whether stress response such as heat stress or exposure to tumor necrosis factor-alpha (TNF-alpha) could induce Hsp70 release from RA fibroblast-like synoviocytes (FLSs) and whether extracellular Hsp70 would stimulate cytokine production in RA FLSs. Cultured FLSs were obtained from patients with RA. The expression of intracellular Hsp70 was studied by Western blot. Hsp70 release and the production of interleukin (IL)-6, IL-8, and IL-10 by RA FLSs were studied by specific enzyme-linked immunosorbent assays. The levels of Toll-like receptor (TLR) 2 and 4 mRNA and protein in FLSs were analyzed using reverse transcription-polymerase chain reaction and Western blotting. Treatment with sublethal heat shock or TNF-alpha results in the up-regulation of intracellular Hsp70 in FLSs and Hsp70 release from RA FLSs. In vitro studies show that extracellular Hsp70 can induce anti-inflammatory cytokine IL-10 production in FLSs. The mRNA and protein expression of TLR2 and TLR4 was demonstrated in FLSs, and TLR4 blocking abrogated the up-regulatory effects of Hsp70 on IL-10 production. Thus, these results lend support to the hypothesis that Hsp70 is actively released from FLSs in response to heat shock or TNF-alpha and Hsp70 may be a major paracrine/autocrine inducer of IL-10 production in FLSs via TLR4.