We investigated the structural basis of calmodulin (CaM)-mediated regulation of the skeletal muscle ryanodine receptor (RyR1), a calcium channel that plays a key role in excitation-contraction coupling in muscle cells. In order to understand the complex interaction of CaM with this receptor, we pursued NMR and X-ray crystallographic studies of CaM/RyR1 peptide complexes both in the presence and in the absence of calcium. We have determined the 2.0 A crystal structure of Ca2+CaM in complex with a 30-residue peptide corresponding to the binding region for CaM on RyR1 (residues 3614 to 3643). The structure reveals that hydrophobic anchor residues in the target arranged in a novel '1-17' spacing allow each calmodulin lobe to interact with the peptide independently. Solution NMR 15N relaxation measurements and residual dipolar couplings confirm the structure of each calmodulin lobe and show that the complex undergoes segmental domain motion. Fluorescence measurements indicate that CaM binds with both domains to the 3614-3643 peptide, whereas if the second anchor is unavailable, CaM can bind without collapsing on the target. The independence of the two lobes of calmodulin offers a structural explanation for how other domains may compete for binding to this region to regulate the channel. NMR studies of these interaction partners indicate that the RyR1 target binds to only the C-lobe of CaM at low calcium concentrations, similar to other ion channels whose activity is modulated by CaM. The conformation of the C-lobe in the calcium-free complex closely resembles the one seen in the crystal structure of the calcium-loaded complex, suggesting that binding of the RyR1 peptide locks the C-domain of CaM in a conformation similar to that of the calcium-loaded protein. Comparison of the CaM/peptide complexes at low and high calcium concentrations provides a model for how CaM interacts with this region of RyR1: the C-lobe is constitutively tethered to the 3614-3643 target and is calcium-loaded even at low, resting calcium levels, whereas high calcium induces the N-lobe to bind to this region. In this way, the N-lobe of CaM acts as a Ca2+ sensor for RyR1 by switching between different binding sites on the receptor.