Rat β-parvalbumin (β-PV) and chicken parvalbumin 3 (CPV3) exhibit diminished Ca(2+) affinity. Their sequences， 70% identical， are unusual in that serine replaces the consensus residue， valine， at position 33. Reasoning that the substitution of a compact， polar hydroxymethyl moiety for a bulky， apolar isopropyl group might contribute to the attenuated Ca(2+) affinities， we have characterized the S33V variants of both proteins. The impact of the mutation in CPV3 differs decidedly from that in rat β. Whereas replacement of S33 by valine in CPV3 causes a substantial increase in the solvent-accessible apolar surface in the Ca(2+)-free protein， the mutation evidently decreases the exposed apolar surface area in rat β. Although the mutation has a minimal effect on divalent ion affinity in both proteins， the ΔΔH and -TΔΔS changes for Ca(2+) binding in CPV3 S33V， but not rat β S33V， are consistent with increased burial of the apolar surface. The influence of the S33V substitution on conformational stability likewise differs for rat β-PV and CPV3. Whereas the stability of the former is virtually unperturbed by the sequence alteration， the latter is destabilized by 0.7 kcal/mol. Moreover， the mutation greatly exacerbates the tendency for CPV3 to aggregate. The concentration and scan rate dependence observed in DSC studies of CPV3 S33V denaturation suggest that unfolding proceeds through an intermediate state that is prone to aggregation. Consistent with this idea， reversible unfolding data， collected at very low protein concentration， likewise indicate that the thermal denaturation is not a two-state process.