Proteins are vital to many functions of biological organisms such as muscle contraction and relaxation. It is known that different isoforms of a certain protein can each play a different physiological function. Consequently, the physiological properties of muscle are highly dependent on the various isoforms of several muscle proteins. We are characterizing one of these muscle proteins, parvalbumin, to understand how it controls numerous processes. Parvalbumin is a myoplasmic protein that aids in relaxation by releasing Mg²⁺ and binding free Ca²⁺, which reduces the intracellular concentration of the ion. Since Ca²⁺ plays a necessary role in contraction by binding to the regulatory protein troponin, a decrease in its intracellular concentration will result in relaxation of the muscle fiber. Although studies have shown differences in the Ca²⁺ dissociation constant (K_d) for parvalbumin from different fish species, there is presently no data on how Ca²⁺ and Mg²⁺ dissociation rates might vary between isoforms of a given fish species. To address this issue, we are studying the Ca²⁺ and Mg²⁺ binding characteristics of parvalbumin isoforms in sheepshead and rainbow trout, fishes known to contain two isoforms of parvalbumin. It has also been shown, that the relative expression of one isoform over the other, has a large effect on relaxation rate in both fish species. Here we report on our isolation and characterization of two isoforms of parvalbumin from sheepshead (Archosargus probatocephalus) and rainbow trout (Oncorhynchus mykiss).