Preconditioning protocols that protect the heart from ischemic injury may aid development of new therapies. However, the temporal window of cardioprotection is limited to a few days after the preconditioning stimulus. Here we report a sustained cardioprotected phenotype in mice expressing a tetracycline transactivator (tTA) transcription factor under the control of the alpha myosin heavy chain (MHC) promoter. MHC-tTA mice were originally designed for tetracycline-regulated gene expression in the heart (tet-system). However, we found that after 45 min. of global ischemia at 37°C, left ventricular developed pressure (LVDP) of Langendorff-perfused MHC-tTA mouse hearts rapidly recovered in 5 minutes to 60% of initial levels, while LVDP of wild type (WT) littermates recovered to only 10% of initial. Improved post-ischemic recovery of function for MHC-tTA hearts was associated with a 50% decrease of infarct size and a significantly smaller release of lactate dehydrogenase to the coronary effluent. Improved post-ischemic recovery was not attributable to differences in coronary flow which was similar for WT- and MHC-tTA hearts during recovery. Moreover, improved post-ischemic recovery of MHC-tTA hearts was not abolished by inhibitors of classical cardioprotective effectors (mitochondrial K_ATP channels, PKC, or adenosine receptors) suggesting a novel mechanism. Finally, the tetracycline analog doxycycline (which inhibits binding of tTA to DNA) did not abolish improved recovery for MHC-tTA hearts. The sustained cardioprotected phenotype of MHC-tTA hearts may have implications for developing new therapies to minimize cardiac ischemic injury. Furthermore, investigations of cardioprotection using the tet-system may be aberrantly influenced by sustained preconditioning induced by cardiac transgenesis with tTA.