We describe a method for the removal of endogenous troponin (Tn) complex from bundles of detergent-treated cardiac fibers. After 70 min treatment with cTnT-cTnI most of the endogenous Tn complex was removed from fiber bundles. Complete reconstitution of the Tn complex was achieved by reconstituting with cardiac troponin C (cTnC) in fully relaxing conditions. Ca2+-dependent maximum force of the fibers treated with cTnT-cTnI or cTnT-cTnI33–211, which was used to aid in the visualization of the troponin exchange, decreased to 85–90% of the force developed by fibers before the treatment. SDS-PAGE analysis of the cTnT-cTnI33–211 and the cTnT77–289-cTnI33–211 treated fiber bundles demonstrated that 70–80% of the endogenous Tn subunits were removed. After reconstitution with cTnC, approximately 80–85% of the Ca2+-regulated force was restored in cTnT-cTnI/cTnI33–211 treated fibers. Our results demonstrate that by minimizing the prolonged exposure of skinned cardiac fiber bundles to rigor conditions, successful exchange of all three subunits of the Tn complex can be accomplished with minimal loss of function.