In the rabbit heart, multiple isoforms of cardiac troponin T (cTnT1 through cTnT5, from largest in size to smallest), a protein essential for calcium-regulated myofibrillar ATPase activity, have been identified, and a correlation has been found between these isoforms and myofilament sensitivity to calcium. We have sought to establish the molecular basis of this diversity. Restriction-digest analysis of genomic DNA has indicated that the rabbit cTnT gene is a single-copy gene. cTnT cDNA clones were isolated from cDNA libraries, yielding a consensus sequence for the protein. Newborn rabbit heart cDNAs, obtained using the reverse-transcriptase polymerase chain reaction (RT-PCR), were amplified using primers derived from this cDNA. Three full-length cDNAs that differed by the inclusion or exclusion of three short nucleotide sequences within the cDNAs were obtained. Amplification in the 5′ half of the cDNAs confirmed that multiple cTnT products arose because of the variable inclusion of an 18- and a 30-nt sequence. The 30-nt sequence has homology with previously described alternatively spliced exons in rat and chicken cTnT, whereas the 18-nt sequence has not been described previously. RT-PCR in the 3′ half of the cDNAs confirmed an additional region of heterogeneity: the presence, in part or in full, or absence of a 9-nt region, which matches the alternatively spliced exon 12 described for rat cTnT. In vitro transcription and translation of four cDNA clones containing both the 18- and 30-nt sequences, the 30-nt sequence, the 18-nt sequence, or neither generated protein isoforms that comigrated with cTnT1, cTnT2, cTnT3, and cTnT4, respectively. Polyclonal antisera, raised against the peptide encoded by the 30-nt region, reacted only with cTnT1 and cTnT2. These results demonstrate that alternative independent splicing of two exons in the NH2-terminal half of the cTnT coding region yields four rabbit cTnT isoforms, and an area of heterogeneity in the C-terminal half provides the potential for 12 isoforms. The previously demonstrated positive correlation between the relative amount of cTnT2 and the sensitivity of those myofilaments to calcium suggests that the 10-residue peptide encoded by the 30-nt exon alters thin-filament regulation of contraction.