Myocardium is generally thought to express no more than two isoforms of troponin T (TnT). We have recently reported that TnT purified from rabbit myocardium is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis Into five proteius (TnT1, TnT2, TnT3, TnT4, and TnT5). In this study, these proteins are characterized immunologically and a novel elaborate maturational profile is described. Myocardium was obtained from 23 days of gestation fetal rabbits and 2-day, 6-week, 3-month, and 6-month postnatal rabbits. The major species in the adult myocardium, TnT4, was identified on sodium dodecyl sulfate-polyacrylamide gels and excised. The protein was electroeluted and purified. An amino acid microsequence of a cleaved fragment of this protein was found to be virtually identical to residues 86-99 from adult rabbit cardiac TnT. The protein, TnT1, was used to raise a polyclonal antibody. This antibody recognized all five isoforms from purified cardiac TnT, but none of the TnT isoforms from fast skeletal muscle. A monoclonal antibody, Mab JLT-12, raised against a highly conserved epitope of rabbit fast skeletal muscle, recognized all five cardiac as well as five skeletal muscle isoforms. Western blots performed on intact myocardial preparations demonstrated that TnT1the cardiac isoform with the slowest electrophoretic mobility, was expressed prominently in the immature hearts, in addition to TnT2, TnT3, and TnT4, but TnT1was not evident in the 3-month and 6-month postnatal hearts. The expression of TnT2also decreased with maturation. Thus, the number of TnT isoforms expressed in the rabbit decreases with maturation. These results demonstrate a greater heterogeneity of expression of cardiac TnT isoforms than was previously suspected and suggest that all the five identified TnT rabbit cardiac isoforms are derived from a different gene than the fast skeletal muscle TnT gene. Sequencing of the rabbit cardiac TnT gene(s) and complementary DNA analysis are needed to establish fully the molecular basis of these five cardiac TnT isoforms.