DNA photolyases are repair enzymes which split (repair) UV-induced cyclobutane DNA lesions. Critical steps in the light-driven repair reaction are the absorption of light by a deazaflavin or methenyl tetrahydrofolate cofactor and the transfer of the excitation energy to a reduced and deprotonated FADH(-) cofactor, which initiates an electron transfer to the dimer lesion. Although most efficient energy transfer requires a close cofactor arrangement, there is a separation of > 17 Angstrom between the cofactors in photolyases. To determine the effect of the large cofactor distance on the repair efficiency, a systematic study with model compounds was performed. A series of compounds were synthesized which contain a model DNA lesion covalently connected to a flavin and a deazaflavin. While the flavin-dimer lesion distance was kept constant in all model compounds, the flavin-deazaflavin distance was incrementally increased. Investigation of the dimer cleavage efficiency shows that compounds with a large cofactor separation possess a low energy-transfer efficiency but split the dimer most efficiently within a few minutes. Model compounds with a close cofactor orientation feature a highly efficient energy transfer from the deazaflavin to the flavin. They are, however, unable to perform the repair of the dimer lesion. At very short cofactor distances, the light-driven repair process is fully inhibited. This is explained by a competitive electron transfer between both cofactors, which hinders the electron transfer to the dimer lesion and hence the dimer splitting. The presented data suggest that the large cofactor separation (17 Angstrom) found in photolyases is a critical parameter that determines the DNA repair efficiency by photolyases.