We have demonstrated previously that the STAT-1 transcription factor plays a key role in ischemia/reperfusion (I/R)-induced apoptosis in cardiac myocytes. In the present study we assessed which region of the STAT-1 molecule mediates apoptosis in cardiac myocytes. A STAT-1 construct (amino acid 350-750) lacking the N-terminus could enhance I/R-induced apoptosis in cardiac myocytes. However, a STAT-1 construct, which lacks 60 amino acids at the C-terminus (amino acid 691-750), was ineffective in promoting I/R-induced apoptosis in cardiac myocytes. Furthermore, overexpression of a C-terminal STAT-1 construct (amino acid 691-750) containing the transcriptional activation domain, but not the DNA binding domain, strongly enhanced I/R-induced apoptotic cell death. Cardiac myocytes isolated from mice expressing a truncated C-terminal STAT-1 were more sensitive to I/R-induced cell death. Finally, isolated hearts from these animals exposed to I/R injury had larger infarct size and greater number of TUNEL-positive myocytes than control hearts. These studies demonstrate that the C-terminal transactivation domain of STAT-1 is necessary and sufficient for I/R injury-induced apoptosis in cardiac myocytes.