The local motion of Tyr13 in wild type and mutant calbindin (Mr 8500, 75 amino acids) was investigated by time-resolved fluorescence spectroscopy performed at the MAX synchrotron in Lund, Sweden. Two-dimensional fluorescence spectroscopy (excitation-emission mapping) was used to characterize the emission of Tyr13 against the background of phenylalanine residues in the presence and absence of Ca2+. Local restricted motion of Tyr13 is observed in wild-type calbindin with only minor differences between the Ca2(+)-saturated and Ca2(+)-free forms. In a mutant, where Pro20 is exchanged for Gly and Ala14 and Asn21 are deleted, the local mobility of Tyr13 is enhanced close to values characteristic for free rotational diffusion. An increase of the overall rotational motion in this mutant form by a factor of two and the enhanced local mobility of Tyr13 indicate local and global conformational changes that also affect the Ca2(+)-binding properties. Tyr13 occurs in two isomeric species differing in lifetime of the excited state; the major species is populated to 85-90%.