Osteopontin is a secreted glycoprotein implicated in a variety of functions, including cell adhesion and migration. Because these functions may be of general importance in the response of tissue to injury, we examined osteopontin expression after experimental cardiac injury and human myocardial infarction. Rat hearts were injured by transdiaphragmatic freeze-thaw and examined from 1 to 28 days after injury. Osteopontin was absent from normal myocardium by immunocytochemistry, Western blotting, and in situ hybridization. On days 1 and 2 after injury, osteopontin mRNA and protein were expressed at high levels by macrophages infiltrating necrotic myocardium. Double labeling with the macrophage marker ED1, however, demonstrated that only a subset of macrophages expressed osteopontin. Western blot analysis showed a single 66-kd band in injured myocardium that was absent from control tissue. Although macrophages remained abundant in the ensuing granulation response and scar tissue formation, the expression of osteopontin was diminished on day 4 and markedly downregulated at 1 and 4 weeks after injury, with only rare cells expressing the message or protein. In a human heart with an 8-day-old myocardial infarct, there was abundant expression of osteopontin mRNA and protein in macrophages within the necrotic and granulation tissue. Transient expression of osteopontin was also observed in a subset of macrophages infiltrating lung, skin, and skeletal muscle injured during the experiment, indicating the response was not limited to the heart. Thus, synthesis of osteopontin by macrophages appears to be a generalized response in the reaction to tissue injury. Although macrophages persist in these lesions, osteopontin is dramatically downregulated as healing proceeds. These results provide the first evidence that osteopontin may be important in healing after tissue injury, possibly in cellular adhesion, chemotaxis, and/or phagocytosis.