Several cell types within ventricular muscle express one or more of the three isoforms (neural NOS, or NOS1; inducible NOS, or NOS2; endothelial NOS, or NOS3) of nitric oxide synthase identified thus far. In cardiac microvascular endothelial cells and myocytes, the expression of NOS2 in response to exposure to specific combinations of inflammatory cytokines is differentially regulated through cell-specific second messenger pathways regulating transcriptional activators. Even though NOS3 is constitutively expressed in endothelial cells and cardiac myocytes from both rodent and human species, NOS3 transcript abundance in endothelial cells from large vessels can also be modulated by shear stress, exercise, estrogens and cytokines, and in ventricular myocytes by cAMP. NOS2 and NOS3 activities are also regulated post-translationally in these cells through alterations in endogenous levels of tetrahydrobiopterin or transmembrane L-arginine transport. In addition, the activity of the constitutive isoforms is tightly regulated by agonist-evoked changes in intracellular calcium. NO produced by NOS3 and NOS2 attenuates the contractile responsiveness of cardiac myocytes to beta-adrenergic stimulation, and NOS3 mediates the parasympathetic regulation of L-type calcium current in sino-atrial and ventricular myocytes. NOS1 is expressed in orthosympathetic nerve terminals where it regulates the release of catecholamines in the heart. In view of the apparent multiplicity of isoforms of NOS expressed in cardiac muscle, a tight molecular regulation of their expression and activity at the transcriptional and posttranscriptional level may be needed to coordinate the many physiological roles of NO in the heart.