Background: D-dimer has been used to rule out pulmonary embolism (PE). Based on previous reports of decreased concentrations of coagulation factor XIII (FXIII) in venous thromboembolism, and no change in FXIII concentration in patients with acute cardiovascular disease, we evaluated the benefit of simultaneously measuring D-dimer and FXIII concentrations for diagnosing PE. Methods: In this prospective single-center study, we enrolled 209 patients initially suspected of having PE, and measured their D-dimer and FXIII concentrations. Forty-one patients were diagnosed with PE and 168 with other final diagnoses, including acute coronary syndrome (ACS); aortic dissection (AD); spontaneous pneumothorax (SP); other respiratory, heart, digestive and nervous diseases; and uncertain diagnoses. Results: Patients with PE had significantly higher D-dimer and lower FXIII concentrations than did patients without PE. Combined D-dimer and FXIII measurements provided a higher positive predictive value (76.6%) for PE than single tests, especially in patients with Wells score >4.0 (89.3%). Specifically, patients with AD or ACS showed higher FXIII concentrations and mean platelet volumes than did patients with PE or SP, and patients with PE and AD had higher D-dimer concentrations than did other patients. At the thresholds of 69.0% for FXIII and 1.10 mu g/mL for D-dimer, 123/151 patients (81.5%) with serious diseases (PE, AD, ACS and SP) were correctly distinguished. Conclusions: Combined measurement of D-dimer and FXIII helps distinguish PE from serious diseases with similar symptoms and appears to relate to increased FXIII release from active platelets in cardiovascular disease.