The accommodation of Mg2+ in the N-terminal domain of calmodulin was followed through amide 1H and 15N chemical shifts and line widths in heteronuclear single-quantum coherence spectroscopy NMR spectra. Mg2+ binds sequentially to the two Ca2+-binding loops in this domain, with affinities such that nearly half of the loops would be occupied by Mg2+ in resting eukaryotic cells. Mg2+ binding seems to occur without ligation to the residue in the 12th loop position, previously proven largely responsible for the major rearrangements induced by binding of the larger Ca2+. Consequently, smaller Mg2+-induced structural changes are indicated throughout the protein. The two Ca2+-binding loops have different Mg2+ binding characteristics. Ligands in the N-terminal loop I are better positioned for cation binding, resulting in higher affinity and slower binding kinetics compared with the C-terminal loop II (k(off) = 380 \ 40 s-1 compared with 10,000 s-1 at 25 \C). The Mg2+-saturated loop II undergoes conformational exchange on the 100-$s time scale. Available data suggest that this exchange occurs between a conformation providing a ligand geometry optimized for Mg2+ binding and a conformation more similar to that of the empty loop.