The interactions of various pyrimidines (1,3-dimethylthymine, DMT, 1,3-bis(N-4,N-4-dimethylcytosin-1-yl)propane, DMC) and their corresponding cis-syn cyclobutane dimers (DMTD and DMCD) with a series of excited-state electron donors were examined with the goal of understanding me energetics and mechanism of UV repair by DNA photolyase. For each substrate there is a good correlation between the excited state oxidation potential (E(ox)*) and the quenching rate constant (k(q)). The value for k(q) increases as E(ox)* becomes more negative, asymptotically approaching a value that is at or below the solvent diffusion limit. These data all showed good fits to the Rehm-Weller equation. Reduction potentials for each of the substrates could be extracted from this analysis : -2.20 V (vs SCE) for DMTD; -2.14 V for DMT; -2.17 V for DMCD; and -2.16 for DMC. These values show that the initial electron transfer step in the photolyase mechanism is exergonic by ca. 10-15 kcal/mol. Thus these data support the reductive electron transfer mechanism for DNA photolyases proposed by Jorns et al. (J. Biol. Chem. 1987, 262, 486-491).