Nitric oxide (NO) is a potent paracrine factor that affects myocardial contractility and growth and that may be implicated in reperfusion injury, endothelial dysfunction, and allograft rejection. While both endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) isoforms have been detected in non-human mammalian tissues, the transcription, translation, and localization of both NOS isoforms in normal and failing human atrial and ventricular myocardium remains controversial. The present study was designed to investigate the genetic expression and localization of eNOS and iNOS in cardiac tissue obtained during cardiac transplantation in the presence (recipient) and absence (donor) of congestive heart failure (CHF). Atrial and ventricular tissue were obtained from 14 donors and 14 end-stage CHF patients undergoing cardiac transplantation. Northern blot analysis and in situ hybridization demonstrated similar levels of both eNOS and iNOS mRNA in atrial and ventricular tissue from normal and failing human hearts. Western blot analysis demonstrated that both eNOS and iNOS are present in human myocardium with no crossreactivity between eNOS and iNOS antibodies. Positive immunohistochemical staining was observed within the cytoplasm of atrial and ventricular cardiomyocytes. No significant differences in eNOS and iNOS immunoreactivity were detected. Both eNOS and iNOS activity was present at similar levels in normal and failing atrial and ventricular myocardium. In conclusion, the present studies demonstrate, for the first time, the integrated transcription, translation, and distribution of both eNOS and iNOS isoforms in normal and failing atrial and ventricular human myocardium.