High-mobility group box chromosomal protein 1 (HMGB1) is a protein with both intranuclear functions and extracellular cytokine-like effects. In this report, we study possible candidate receptors for HMGB1 on macrophages (Mφ) and define pathways activated by HMGB1 binding. Bone marrow Mφ were prepared from Dark Agouti (DA) rats and stimulated in vitro with HMGB1. The kinetics of tumour necrosis factor (TNF) production, NO production, activation of p38 mitogen-activated protein kinase (MAPK), p44/42 MAPK- and SAPK/JNK-signalling pathways, nuclear translocation of nuclear factor kappa B (NF-κB) and HMGB1-induced upregulation of major histocompatibility complex (MHC) class II and CD86 were analysed. Mφ from interleukin (IL)-1 receptor type I–/–, Toll-like receptor 2 (TLR2–/–) and RAGE–/– mice were used to investigate the role of these receptors in HMGB1 signalling. HMGB1 induced TNF and NO production by Mφ, phosphorylation of all investigated MAP kinase pathways and NF-κB translocation, and expression of MHC class II was increased. Mφ from RAGE–/– mice produced significantly lower amounts of TNF, IL-1β and IL-6, while IL-1RI–/– and TLR2–/– Mφ produced cytokine levels comparable with wildtype controls in response to HMGB1 stimulation. We conclude that HMGB1 has the potential to induce a proinflammatory phenotype in Mφ, with RAGE as the major activation-inducing receptor.