A novel method for initiation of intramolecular electron transfer reactions in cytochrome c is reported. The method is based on photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS) by the third harmonic frequency of a Nd:YAG laser (355 nm), the reaction that generates the low-potential triplet state of the dye with high quantum efficiency. TUPS derivatives of horse heart cytochrome c singly labeled at specific lysine residues were prepared and purified to homogeneity by ion-exchange high- pressure liquid chromatography. Eight derivatives were characterized by determination of the location of the modification, reduction potentials, and measurement of enzymatic activity with cytochrome oxidase. Transient absorption spectroscopy was used to directly measure the rate constants for the electron transfer reaction from the photoexcited triplet state of TUPS to the oxidized heme group and the back reaction from the ferrous heme to the oxidized dye. For all singly labeled derivatives, the rate constants for heme reduction were 1 or 2 orders of magnitude larger than for its reoxidation, consistent with the greater thermodynamic driving force for the oxidation reaction. Analysis of the variation of electron-transfer rates with the distance separating the dye and the heme reveals a value of coupling decay constant (β) of 0.46 Å-1. Rapid and effective photoreduction of cytochrome c makes it a useful tool for fast initiation of electron transfer in the reductive direction within complexes of cytochrome c with other redox proteins.