Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (alpha and beta isoforms), Baltic cod, and rat (alpha and beta isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike alpha-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 degrees C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca(2+) ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg(2+)- and Na(+)-loaded forms of pike alpha-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg(2+)- and Na(+)-loaded forms of pike alpha-PA), to the more complex mechanisms (Ca(2+)-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca(2+)-loaded pike alpha-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike alpha-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na(+)-bound state), thermophilic (Mg(2+)-form), or even of the hyperthermophilic origin (Ca(2+)-form).