Objectives: Heparin is a highly charged polysaccharide used as an anticoagulant to prevent blood coagulation in patients with presumed myocardial infarction and to prepare heparin plasma samples for laboratory tests. There are conflicting data regarding the effects of heparin on the measurement of cardiac isoforms of troponin I (cTnI) and troponin T (cTnT), which are used for the immunodiagnosis of acute myocardial infarction. In this study, we investigated the influence of heparin on the immunodetection of human cardiac troponins. Methods: Gel filtration (GF) techniques and sandwich fluoroimmunoassay were performed. The regions of сTnI and cTnT that are affected by heparin were investigated with a panel of anti-cTnI and anti-cTnT monoclonal antibodies, specific to different epitopes. Results: Heparin was shown to bind to the human cardiac full-size ternary troponin complex (ITC-complex) and free cTnT, which increased their apparent molecular weights in GF studies. Heparin did not bind to the low molecular weight ITC-complex and to binary cTnI-troponin С complex. We did not detect any sites on cTnI in the ITC-complex that were specifically affected by heparin. In contrast, cTnT regions limited to approximately 69–99, 119–138 and 145–164 amino acid residues (aar) in the ITC-complex and a region that lies approximately between 236 and 255 aar of free cTnT were prone to heparin influence. Conclusions: Heparin binds to the ITC-complex via cTnT, interacting with several sites on the N-terminal and/or central parts of the cTnT molecule, which might influence the immunodetection of analytes in human blood.