The FRQ1 gene is essential for growth of budding yeast and encodes a 190-residue, N-myristoylated (myr) calcium-binding protein. Frq1 belongs to the recoverin/frequenin branch of the EF-hand superfamily and regulates a yeast phosphatidylinositol 4-kinase isoform. Conformational changes in Frq1 due to N-myristoylation and Ca²⁺ binding were assessed by nuclear magnetic resonance (NMR), fluorescence, and equilibrium Ca²⁺-binding measurements. For this purpose, Frq1 and myr-Frq1 were expressed in and purified from Escherichia coli. At saturation, Frq1 bound three Ca²⁺ ions at independent sites, which correspond to the second, third, and fourth EF-hand motifs in the protein. Affinity of the second site (K_d = 10 μM) was much weaker than that of the third and fourth sites (K_d = 0.4 μM). Myr-Frq1 bound Ca²⁺ with a K_dapp of 3 μM and a positive Hill coefficient (n = 1.25), suggesting that the N-myristoyl group confers some degree of cooperativity in Ca²⁺ binding, as seen previously in recoverin. Both the NMR and fluorescence spectra of Frq1 exhibited very large Ca²⁺-dependent differences, indicating major conformational changes induced upon Ca²⁺ binding. Nearly complete sequence-specific NMR assignments were obtained for the entire carboxy-terminal domain (residues K100−I190). Assignments were made for 20% of the residues in the amino-terminal domain; unassigned residues exhibited very broad NMR signals, most likely due to Frq1 dimerization. NMR chemical shifts and nuclear Overhauser effect (NOE) patterns of Ca²⁺-bound Frq1 were very similar to those of Ca²⁺-bound recoverin, suggesting that the overall structure of Frq1 resembles that of recoverin. A model of the three-dimensional structure of Ca²⁺-bound Frq1 is presented based on the NMR data and homology to recoverin. N-myristoylation of Frq1 had little or no effect on its NMR and fluorescence spectra, suggesting that the myristoyl moiety does not significantly alter Frq1 structure. Correspondingly, the NMR chemical shifts for the myristoyl group in both Ca²⁺-free and Ca²⁺-bound myr-Frq1 were nearly identical to those of free myristate in solution, indicating that the fatty acyl chain is solvent-exposed and not sequestered within the hydrophobic core of the protein, unlike the myristoyl group in Ca²⁺-free recoverin. Subcellular fractionation experiments showed that both the N-myristoyl group and Ca²⁺-binding contribute to the ability of Frq1 to associate with membranes.