A rapid 30-minute assay of circulating smooth-muscle myosin heavy-chain protein has been developed as a biochemical diagnostic tool for aortic dissection. To determine the sensitivity and specificity of this assay. Cross-sectional study. 8 major cardiovascular centers in Japan. 95 patients with acute aortic dissection, 48 patients with acute myocardial infarction, and 131 healthy volunteers. Levels of circulating smooth-muscle myosin heavy-chain protein. Patients with acute aortic dissection who presented within 3 hours after onset had elevated levels of circulating smooth-muscle myosin heavy-chain protein. In these patients, the assay had a sensitivity of 90.9%, a specificity of 98% compared with healthy volunteers, and a specificity of 83% compared with patients who had acute myocardial infarction; the clinical decision limit was 2.5 microgram/L. All patients with proximal lesions had elevated levels of smooth-muscle myosin heavy-chain protein, and only patients with distal lesions had decreased levels (<2.5 microgram/L). Levels of smooth-muscle myosin heavy-chain protein can be used to diagnose aortic dissection soon after symptom onset. The assay had the greatest diagnostic value in patients with proximal lesions.