To evaluate the performances of a new D-dimer assay, Stratus CS D-dimer (Dade Behring, Newark, Delaware, USA), for the exclusion of venous thromboembolism and to determine the best cut-off values. This test relies on a sandwich enzyme-linked immunosorbent assay with a fluorogenic substrate in a radial partition immunoassay system in a dedicated device. It does not require laboratory staff and the results are available within 18 min. Two hundred and ninety-one frozen samples from patients suspected of either deep venous thrombosis (n = 137) or pulmonary embolism (n = 154) were measured and the results compared with the VIDAS D-dimer New assay. There were 37 deep venous thrombosis (27%) and 49 pulmonary embolism (32%) samples. Receiver–operating characteristic curve analysis indicated that the cut-off value could be set at either 300 or 400 ng/ml Fibrinogen Equivalent Units (FEU). The coefficient of variation determined with a lyophilized low-level control plasma (305 ng/ml FEU) was 6%. With a cut-off at 300 ng/ml FEU, the sensitivity, the specificity and the negative predictive value were 100% [95% confidence interval (CI), 95.8–100], 33.1% (95% CI, 26.8–40) and 100% (95% CI, 94.7–100), respectively. Raising the cut-off to 400 ng/ml FEU, the corresponding figures were 96.5% (95% CI, 90.1–99.3), 46.3% (95% CI, 39.4–53.2) and 96.9% (95% CI, 91.3–99.4), respectively. At 400 ng/ml, three results were false-negative, one with both devices and two with the Stratus D-dimer only. Stratus D-dimer appears to be suitable for the exclusion of venous thromboembolism in the emergency room setting.