The cytosolic Ca²⁺/Mg²⁺ binding protein -Parvalbumin (-Parv) has been shown to accelerate cardiac relaxation; however, beyond an optimal concentration range -Parv can also diminish contractility. Mathematical modeling suggests that increasing Parv Mg²⁺ affinity may lower the effective [Parv] to speed relaxation and thus limit Parv-mediated depressed contraction. Naturally occurring Parv isoforms show divergence in amino acid primary structure (57% homology) and in cation binding affinities, with -Parv isoform having an estimated 16% increase in Mg²⁺ affinity and ~ 200% increase in Ca²⁺ affinity relative to &#945;-Parv. We tested the hypothesis that at the same or lower estimated [Parv], -Parv would more significantly accelerate mechanical relaxation rate relative to -Parv. Dahl salt-sensitive (DS) rats were used as an experimental model of diastolic dysfunction. Adult cardiac myocytes isolated from DS rats had significantly slowed relaxation properties compared to controls [Time from Peak contraction to 50% relaxation 57 ± 2 vs. 49 ± 2 msec, DS vs. control, mean ± SEM, P <0.05], validating this model system. DS cardiac myocytes were subsequently transduced with -Parv or -Parv adenoviral vectors. Upon Parv gene transfer, -Parv caused significantly faster relaxation than -Parv (P <0.05), even though estimated [-Parv] was 10% of [ Parv]. This comparative analysis showing distinct functional outcomes raises the prospect of utilizing naturally occurring Parv variants to address disease-associated slowed cardiac relaxation.