Using a monoclonal antibody (CDC4) that recognizes both the cardiac and slow skeletal isoforms of troponin T in an immunoblotting procedure, the composition of troponin T isoforms in adult and developing skeletal muscles of the rat and human were studied. Two major isoforms of slow troponin T (HS1 and HS2) were detected in all the adult human skeletal muscles investigated. Significant amounts of another isoform (HS3) in addition to HS1 and HS2 were also detectable in a subgroup of these muscles. All the human fetal skeletal muscles at 20 weeks of gestation expressed HS1 and HS2 isoforms but not HS3. The fetal skeletal muscles, also expressed cardiac troponin T in addition. Unlike the human skeletal muscles, only a single isoform of slow troponin T was detected by antibody CDC4 in both the adult and neonatal rat skeletal muscles. The investigation of fetal rat skeletal muscles using the same antibody, however, detected the presence of not only the embryonic cardiac and adult slow skeletal isoforms but also five additional not previously described isoforms (Es1-Es5) of troponin T. These embryonic isoforms, Es1-Es5, were undetectable in the postnatal skeletal muscles although their small amounts could be detected in the neonatal rat hearts. The analysis of individual skeletal muscles from different parts of the body at different stages of fetal development showed marked variations in both the composition of troponin T isoforms and the time sequence of their transitions in each muscle.