AIMS: Pathogenic effects of the endogenous inflammatory mediator high mobility group box protein 1 (HMGB1) have been described in several inflammatory diseases. Recent reports have underlined the importance of post-translational modifications (PTMs) in determination of HMGB1 function and release mechanisms. We investigated the occurrence of PTMs of HMGB1 obtained from synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients. RESULTS: Analyses of 17 JIA patients confirmed high HMGB1 levels in SF. Liquid chromatography tandem mass-spectrometry (LC-MS/MS) analyses of PTMs revealed that total HMGB1 levels were not associated with increased lactate dehydrogenase activity but strongly correlated with nuclear location sequence 2 (NLS2) hyperacetylation, indicating active release of HMGB1. The correlation between total HMGB1 levels and NLS2 hypoacetylation suggests additional, acetylation-independent release mechanisms. Monomethylation of lysine 43 (K43), a proposed neutrophil-specific PTM, was strongly associated with high HMGB1 levels, implying that neutrophils are a source of released HMGB1. Analysis of cysteine redox isoforms, fully reduced HMGB1, disulfide HMGB1, and oxidized HMGB1, revealed that HMGB1 acts as both a chemotactic and a cytokine-inducing mediator. These properties were associated with actively released HMGB1. INNOVATION: This is the first report that characterizes HMGB1-specific PTMs during a chronic inflammatory condition. CONCLUSION: HMGB1 in SF from JIA patients is actively released through both acetylation-dependent and -nondependent manners. The presence of various functional HMGB1 redox isoforms confirms the complexity of their pathogenic role during chronic inflammation. Defining HMGB1 release pathways and redox isoforms is critical for the understanding of the contribution of HMGB1 during inflammatory processes.