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The kinetics of reduction of cytochrome b and cytochrome c1 of yeast Complex III by 5-deazariboflavin semiquinone, generated by laser flash photolysis under anaerobic conditions, have been investigated. The reduction of cytochrome b occurs in two phases with first-order rate constants of 1300 and 670 s-1, whereas the reduction of cytochrome c1 appears as a unique exponential phase with an intermediate value of 800 s-1. Under these experimental conditions, about 50% of cytochrome b is reduced in comparison with cytochrome c1. After photoreduction, the re-oxidation of the cytochromes by internal re-equilibrium occurs in both cases, following pseudo-first-order kinetics at a rate constant of 43 s-1 for cytochrome b and 39 s-1 for cytochrome c1. These results, which agree with the data from the rapid mixing technique (A.-L. Tsai, J.S. Olson, G. Palmer, J. Biol. Chem. 262 (1987) 8677-8684), have implications for the mechanistic understanding of inner Complex III electron transfer. One of the goals of the investigation reported here is to provide direct evidence for the hypothesis of a proton-motive Q cycle for the mechanism of electron transfer in Complex III. Moreover, these results demonstrate the usefulness of laser flash photolysis in studying the redox kinetic properties of mitochondrial Complex III.