BACKGROUND Autoantibodies to cardiac troponins (cTnAAbs) can interfere with the measurement of cardiac troponin I (cTnI) by immunoassays for the diagnosis of myocardial infarction. Therefore, we determined the cTnI binding sites and IgG subclasses of circulating cTnAAbs. METHODS We studied epitope specificity with sandwich-type immunoassays by measuring the recovery of troponin complex added to 10 cTnAAb-negative and 10 cTnAAb-positive sera from healthy volunteers. To study the IgG subclasses, we analyzed admission and 3-month follow-up sera from chest pain patients with a reference assay measuring total IgG (14 cTnAAb negative and 14 cTnAAb positive at 3 months) and with 4 subclass-specific assays measuring exclusively IgG subclasses 1-4. RESULTS Mean recoveries of troponin complex in cTnAAb-positive samples for single cTnI epitopes ranged from 37% to 211%, being lowest for the cTnI midfragment (aa 30-110). However, the lowest sample-specific recoveries, 4%-92%, showed that none of the studied epitopes completely escaped the cTnAAb-related interference. Eight chest pain patients of the cTnAAb-positive group became positive between sampling points, and according to all 5 cTnAAb assays, specific signals were generally higher at follow-up. IgG4, with the highest prevalence, was detected in 68% of samples in the cTnAAb-positive group. CONCLUSIONS IgG subclass studies confirm that cTnAAb formation may be triggered/boosted in acute cardiac events. This new information about the epitope specificity of cTnAAbs should be used to reevaluate existing recommendations regarding use of midfragment epitopes in cTnI assays. To circumvent the negative interference of the highly heterogeneous cTnAAbs, use of 3 or more unconventionally selected epitopes should be considered.