High mobility group box-1 protein (HMGB1), a recently described late-acting cytokine that mediates lethality of sepsis and systemic inflammation, also plays a role in mediating dendritic cell (DC) maturation and activation. The present study was performed to clarify the effects of HMGB1 on splenic DCs and its potential regulating mechanism underlying T-cell-mediated immunity. DCs isolated from the spleens of normal rats were treated with HMGB1 of different dosage (0.1, 1, or 10 microg/mL) for different duration (24, 48, or 72 h). Expressions of co-stimulatory molecules, including CD80, CD86, and MHC-II on DCs surface, and cytokines, including interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, were analyzed to identify DCs maturation and activation. The activated DCs were assessed for their capacity to stimulate the proliferation and differentiation of T cells. Expression of the receptor for advanced glycation end products (RAGE) on DCs and nuclear factor (NF)-kappaB activation in T lymphocytes were also determined. Stimulation with HMGB1 markedly up-regulated the co-stimulatory molecules and cytokines expressions, and they peaked at 48 h when DCs was treated with 1 microg/mL HMGB1. Treatment with anti-RAGE antibody prevented the maturation of DCs. DCs treated with HMGB1 (1 microg/mL for 48 h) promoted T-cell proliferation as well as differentiation, and markedly up-regulated IL-2, IL-2R expression and intranuclear NF-kappaB activation. The results suggested that HMGB1 appear to be a potential immunostimulatory signal that induced DC maturation and T-cell-mediated immunity, and RAGE was a potential receptor associated with maturation and differentiation of DCs. Moreover, HMGB1 might have a dual regulatory effect on immune functions of DCs varying with different concentration and stimulation time.