Cytochrome c (cyt c) was reduced by a tyrosine-containing peptide, tyrosyltyrosylphenylalanine (TyrTyrPhe), at pH 6.0-8.0, while tyrosinol or tyrosyltyrosine (TyrTyr) could not reduce cyt c effectively under the same condition. Cyt c was reduced at high peptide concentration, whereas the reaction did not occur effectively at low concentration. The reaction rate varied with time owing to a decrease in the TyrTyrPhe concentration and the production of tyrosine derivatives during the reaction. The initial rate constants were 2.4 x 10(-4) and 8.1 x 10(-4) s(-1) at pH 7.0 and 8.0, respectively, for the reaction with 1.0 mM TyrTyrPhe in 10 mM phosphate buffer at 15 degrees C. The reciprocal initial rate constant (1/k(int)) increased linearly against the reciprocal peptide concentration and against the linear proton concentration, whereas logk(int) decreased linearly against the root of the ionic strength. These results show that deprotonated (TyrTyrPhe)(-), presumably deprotonated at a tyrosine site, reduces cyt c by formation of an electrostatic complex. No significant difference in the reaction rate was observed between the reaction under nitrogen and oxygen atmospheres. From the matrix-assisted laser desorption ionization time-of-flight mass spectra of the reaction products, formation of a quinone and other tyrosine derivatives of the peptide was supported. These products should have been produced from a tyrosyl radical. We interpret the results that a cyt c(ox)/(TyrTyrPhe)(-)right harpoon over left harpooncyt c(red)/(TyrTyrPhe)(*) equilibrium is formed, which is usually shifted to the left. This equilibrium may shift to the right by reaction of the produced tyrosyl radical with the tyrosine sites of unreacted TyrTyrPhe peptides.