Hydrogen sulfide (H(2)S) has emerged as an important gasotransmitter, offering protection against ischemia-reperfusion damage to the heart. Cystathionine gamma-lyase (CSE) is believed to be the major H(2)S-generating enzyme in the heart. Quite contrary to the general contemplation, CSE protein in cardiac tissues has not been convincingly detected and it has become an issue of controversy. In the present study, we isolated cardiac tissues from wild type (WT) and CSE knockout mice or the rat. CSE expression at transcriptional and translational levels were assayed by RT-PCR and Western Blotting with five different antibodies (four commercial products and one homemade), respectively. Cardiac H(2)S production rate was also examined. Our data validated the expression of CSE mRNA in the heart of WT mice or rats, not in CSE KO mice. Using all 5 different anti-CSE antibodies, we could not detect CSE proteins in mouse or rat cardiac tissues or in cultured rat cardiomyocytes. On the other hand, CSE protein was detectable in liver tissues from WT mice with the expected molecular mass of 43.6 kDa. H(2)S production rate of heart tissues in CSE KO mice was significantly decreased compared with that in WT mice. In the presence of an CSE inhibitor, D,L-propargylglycine, H(2)S production rate of heart tissues from WT mice was inhibited by approximately 80%. It appears that CSE mediates mostly endogenous H(2)S production in heart tissues. However, the available anti-CSE antibodies could not detect CSE proteins in rat and mouse heart tissues or rat cardiomyocytes.