The discovery that plants contain multiple calmodulin (CaM) isoforms of variable sequence identity to animal CaM suggested an additional level of sophistication in the intracellular role of calcium regulation in plants. Past research has focused on the ability of conserved or divergent plant CaM isoforms to activate both mammalian and plant protein targets. At present, however, not much is known about how these isoforms respond to the signal of an increased cytosolic calcium concentration. Here, using isothermal titration calorimetry and NMR spectroscopy, we investigated the calcium binding properties of a conserved (CaM1) and a divergent (CaM4) CaM isoform from soybean (Glycine max). Both isoforms bind calcium with a semisequential pathway that favors the calcium binding EF-hands of the C-terminal lobe over those of the N-terminal lobe. From the measured dissociation constants, CaM4 binds calcium with a threefold greater affinity than CaM1 (K(d,Ca,mean) of 5.0 versus 14.9 μM) but has a significantly reduced selectivity against the chemically similar magnesium cation that binds preferentially to EF-hand I of both isoforms. The implications of a potential magnesium/calcium competition on the activation of CaM1 and CaM4 are discussed in context with their ability to respond to stimulus-specific calcium signatures and their known physiological roles.