Excitation-contraction (EC) coupling is driven by an ion-channel-mediated calcium cycle that produces myofilament contraction and relaxation. Even though nitric oxide synthases (NOS) were definitively described within the heart a decade ago, the role that nitric oxide (NO) plays in cardiac regulation remains highly controversial. There is a growing consensus, however, that NO modulates the activity of several key calcium channels involved in EC coupling as well as mitochondrial respiratory complexes. To accomplish this regulation, different NOS isoforms are spatially confined in distinct cellular microdomains involved in EC coupling. Specifically, NOS1 localizes to the sarcoplasmic reticulum (SR) in proximity to the ryanodine receptor (RYR) and the SR Ca(2+) ATPase (SERCA2a), and NOS3 is found in sarcolemmal caveolae compartmentalized with cell-surface receptors and the L-type Ca(2+) channel. NO also participates in mitochondrial respiration, the process that fuels EC coupling, and either NOS1 or 3 resides within cardiac mitochondria. Here, we review the biochemical and cellular mechanisms whereby NO influences EC coupling. There is accumulating evidence that NO participates in all aspects of EC coupling, including receptor signal transduction, L-type Ca(2+)-channel activity, SR calcium release through the RYR, and mitochondrial respiration.