The aim of this study was to measure the direct release of nitric oxide (NO) from the porcine mitral valve using a NO microelectrode. Furthermore, the expression and localization of endothelial nitric oxide synthase (eNOS) in the mitral valve was studied using immunohistochemistry, Western blotting and RT-PCR. Results show that bradykinin increases NO release from mitral valves (¿Bradykinin: 33.71 ± 10.41 nM NO, P < 0.001, n = 10), whereas N-nitro-L-arginine methyl esther (L-NAME) decreases NO release when compared with basal level (¿L-NAME: 82.69 ± 15.66 nM NO, P < 0.005, n = 4). Both protein and mRNA expression of eNOS in mitral valves and in isolated valvular endothelial cells suggest that the NO release is mainly associated with the mitral valve endothelium. It is concluded that direct NO release from porcine mitral valves coincides with eNOS expression. This study documents useful techniques for investigations into the role of local NO release in mitral valve diseases.