Abstract By specific antiboby screening of a λZAPII expression library, we identified a class of cDNAs derived from mouse cardiac troponin T (TnT) mRNA with long 5′-ends containing the -30 TATA sequence of the promoter. These long mRNAs indicated the presence of a novel upstream transcription initiation site in the cardiac TnT gene. Primer extension mapping of the mouse cardiac TnT mRNA confirmed an alternative transcription initiation site 55-base pairs initiation site. The upstream initiation site was found significantly more active in the embryonic/neonatal hearts. Genomic cloning and sequencing further revealed an 1.5-kb 5′-upstream sequence of the mouse cardiac TnT gene and relationships between the promoter regulatory elements and the alternative transcription initiation sites.