The isoform-specific assembly of cardiac and skeletal muscle troponin-I (CTnI and FTnI, respectively) on to myofibrils (MFs) was investigated. Epitope tagging was used to monitor the intracellular localization of exogenously introduced constructs to myofibrillar structures in cultured chicken cardiac and fast skeletal (breast) muscle cells. Exogenous CTnI and FTnI were incorporated into endogenous MFs of cardiac and breast muscle cells with high affinity, respectively. In the case of CTnI and FTnI with breast and cardiac muscle cells respectively, CTnI was not incorporated into breast MFs but FTnI was assembled on to cardiac MFs. To determine which portion of TnI is responsible for incorporation into these MFs, we constructed chimeric TnIs with the head and tail of CTnI replaced by those of FTnI. The behaviour of these chimeras depends on the tail of TnIs. These results suggest that the tail regions of TnIs bind to cardiac and breast MFs, and that this affinity of TnI tails is responsible for the assembly of FTnI on to cardiac MFs.