By using a human cell-free system capable of nucleotide excision repair, a synthetic substrate consisting of a plasmid containing four thymidine dimers at unique locations, and deoxyribonucleoside 5'-[alpha-thio]triphosphates for repair synthesis, we obtained DNA fragments containing repair patches with phosphorothioate linkages. Based on the resistance of these linkages to digestion by exonuclease III and their sensitivity to cleavage by I2, we were able to delineate the borders of the repair patch to single-nucleotide resolution and found an asymmetric patch with sharp boundaries. That the repair patch was produced by filling in a gap generated by an excision nuclease and not by nick-translation was confirmed by the finding that the thymidine dimer was released in a 27- to 29-nucleotide oligomer.