In critically ill patients, either arterial or venous blood is usually available for sampling and measurement of basic coagulation parameters. The aim of this study was to examine whether in these patients the values of coagulation parameters differ significantly with respect to the source of the blood samples. In a group of 44 patients with severe sepsis, we compared the values of coagulation, thromboelastography and selected hematological parameters between the arterial and venous blood. In most of the investigated parameters (international normalized ratio, activated partial thromboplastin time, fibrinogen concentration, erythrocyte count, leukocyte and platelet count, hemoglobin level and thromboelastography parameters), we did not find significant differences (P > 0.1). However, we found a significantly lower antithrombin activity and a significantly higher D-dimer concentration in venous blood compared to arterial blood (P < 0.05). This could be associated with increased consumption of antithrombin and generation of D-dimer as a consequence of microthrombi formation in the capillaries. We therefore conclude that for the purpose of assessment of coagulation status in septic patients, arterial and venous blood cannot be treated as equivalent.