Abstract The 7 F 0 a ̊ 5 D 0 transition of Eu 3+ is used to investigate the metal ion binding sites of five parvalbumin isotypes. Comparisons are based on the resolution of the Eu 3+ excitation spectrum with a computer program using the Marquardt nonlinear regression algorithm. The Lorentzian-Gaussian product function provides the most effective approximation to the shape of the peaks, the positions of which were found to be related to differences in the amino acid residues at the binding sites. Below pH 6, spectra consist of two peaks near 579 nm, but as the pH is raised, the two peaks gradually diminish and two new, much broader peaks appear at 577 and 578 nm. The lower wavelength peak, at low pH, and the peak at 577 nm, at high pH, are assigned to the CD site. The two sites have nearly equal affinities for Eu 3+ except in the northern pike III (p I 5.0) isotype. The excitation spectra are compared to that of the carp III (p I = 4.25) parvalbumin for which the complete crystal structure is available. This structure forms the basis for the molecular modeling studies of the altered binding sites. Preliminary results are presented regarding differences in solvent exposure of the CD and EF sites based on collision-induced energy transfer to [Co(NH 3) 6] 3+.