Photolyases are ancient enzymes that harvest sunlight to repair DNA pyrimidine lesions such as pyrimidine(6-pyrimidine(6-4)pyrimidone and cyclobutane dimers. Particularly, (6-4) photolyase ((6-4)PHR) plays an important role in maintaining genetic integrity by repairing thymine(6-4)thymine (T(6-4)T) and thymine(6-4)cytosine (T(6-4)C) photolesions. The majority of 6-4)PHR studies have been performed on the basis of the former's activity and assuming the equivalence of the two repair mechanisms, although the latter's activity remains poorly studied. Here, we describe investigations of the repair process of the T(64)C dimer using several computational methods from molecular dynamics (MD) simulations to large quantum mechanical/ molecular mechanical approaches. Two possible mechanisms, the historically proposed azetidine four-member ring intermediate and the free NH3 formation pathways, were considered. The MD results predicted that important active site histidine residues employed for the repair of the T(6-4)C dimer have protonation states similar to those seen in the (6-4)PHR/T(6-4)T complex. More importantly, despite chemical differences between the two substrates, a similar repair mechanism was identified: His365 protonates NH2, resulting in formation/activation mechanism of a free NH3, inducing NH2 transfer to the 5' base, and ultimately leading to pyrimidine restoration. This reaction is thermodynamically favorable with a rate-limiting barrier of 20.4 kcal mol(-1). In contrast, the azetidine intermediate is unfeasible, possessing an energy barrier of 60 kcal mol(-1) ; this barrier is similar to that predicted for the oxetane intermediate in T(6-4)T repair. Although both substrates are repaired with comparable quantum yields, the reactive complex in T(6-4)C was shown to be a 3' base radical with a lower driving force for back electron transfer combined with higher energy barrier for catalysis. These results showed the similarity in the general repair mechanisms between the two substrates while emphasizing differences in the electron dynamics in the repair cycle.