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A mutated form of troponin c from chicken skeletal muscle was used as a spectral probe for monitoring the Ca2+-induced structrural transition of the regulatory N domain. Phe-29 at the COOH-terminal fragment of the A helix near the Ca2+-binding loop of site I was replaced by tryptophan (trp) which elicited unambiguous spectral features owing to the absence of other trp or tyrosine. Alteration in spectral features was restricted to Ca2+-binding in the N domain conformational transition. The spectral feature of the probe mutant was also applied to the analysis of Ca2+-binding affinities of five double mutants wherein nonpolar amino acids were replaced by polar residues.