In the presence of glycosaminoglycans, thrombin is rapidly inactivated by two natural inhibitors secreted from liver: antithrombin (AT) is presumed to be the principal thrombin inhibitor in circulating blood, while for heparin cofactor II (HCII), a role outside circulation has been proposed. In this study, we show that HCII and AT differ with respect to their association with human tissues. Aside from brain, each of these inhibitors was found in sodium dodecyl sulphate (SDS) soluble extracts of various human organs, with a preponderance of HCII in placenta. AT levels, however, predominated in liver. Compared to plasma, the beta-variant of AT was found to be strongly enriched in human organs, while tissue-resident HCII did not differ in its electrophoretic mobility from the circulating form. In placenta, comparable amounts of HCII/thrombin and AT/thrombin complexes were detected, indicating that HCII may exert a thrombin regulating role in that organ under conditions of tissue or blood vessel damage. Transcripts coding for HCII and AT were detected in all tissues examined. The low levels of their mRNAs suggest that most of the tissue-associated thrombin inhibitor molecules originate from circulation and are retained in organs, possibly by specific receptors. The differential presence of HCII and AT in organs is in accordance with individual physiological roles of these inhibitors.