HMGB1 is a highly conserved nuclear protein that displays important biological activities inside as well as outside the cell and serves as a prototypic alarmin to activate innate immunity. The translocation of HMGB1 from inside to outside the cell occurs with cell activation as well as cell death, including apoptosis. Apoptosis is also a setting for the release of cellular microparticles (MPs), which are small membrane-bound vesicles that represent an important source of extracellular nuclear molecules. To investigate whether HMGB1 released from cells during apoptosis is also present on MPs, we determined the presence of HMGB1 on particles released from Jurkat and HL-60 cells induced to undergo apoptosis in vitro by treatment with either etoposide or staurosporine; MPs released from cells undergoing necrosis by freeze-thaw were also characterized. As shown by both Western blot analysis and flow cytometry, MPs from apoptotic cells contain HMGB1, with binding by antibodies indicating an accessible location in the particle structure. These results indicate that HMGB1, like other nuclear molecules, can translocate into MPs during apoptosis and demonstrate another biochemical form of this molecule that may be immunologically active.