Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical for the resolution of inflammation. Plasma and tissue levels of plasminogen activator inhibitor-1 (PAI-1), an inhibitor of fibrinolysis, are elevated in inflammatory conditions, including sepsis and acute lung injury, in which activated neutrophils accumulate in tissues and contribute to organ dysfunction. In this study, we explored the potential involvement of PAI-1 in modulating neutrophil efferocytosis. We found enhanced phagocytosis of viable PAI-1 deficient (PAI-1-/-) and of wild-type neutrophils treated with anti-PAI-1 antibodies. PAI-1 levels were decreased on the surface of apoptotic neutrophils and the enhanced phagocytosis of apoptotic wild-type neutrophils or of viable PAI-1-/- neutrophils was diminished by preincubation with PAI-1. The increased phagocytosis associated with PAI-1 deficiency or blockade depended on both the lipoprotein receptor-related protein (LRP) and its ligand, calreticulin (CRT), because the LRP-mediated increase in phagocytosis of viable neutrophils induced by blockade of CD 47 was abrogated by PAI-1. CRT levels are increased on viable PAI-1 -/- neutrophils. While CRT colocalizes with PAI-1 on viable neutrophils, markedly diminished colocalization of PAI-1 and CRT was present on apoptotic neutrophils. Our data therefore indicate that PAI-1 serves as a novel "don't eat me" signal for viable and apoptotic neutrophils. © 2008 by The National Academy of Sciences of the USA.