The removal by EDTA of Ca2+ from the two-tryptophan-containing metalloproteinase isolated from Staphylococcus aureus leads to an increase in its intrinsic fluorescence intensity. Based on acrylamide fluorescence quenching results, analyzed by the non-linear least-squares method, we have shown that this protein molecule undergoes irreversible conformational change upon removal of Ca2+, which include the exposure to the solvent of buried tryptophan residues. Steady-state fluorescence anisotropy measurements indicate that the loss of Ca2+ leads to a significant increase in internal mobility of previously buried tryptophan residues.