The cis,syn and (6-4) products of dipyrimidine sites are the major mutagenic and lethal UV photoproducts in DNA. To investigate their relative susceptibilities to repair and other factors such as sequence context and lesions in the complementary strand that might influence repair efficiencies, we constructed 49-mer duplexes containing site-specific photoproducts of thymidylyl(3',5')thymidine sites at central locations. Using these substrates, we measured the binding of Escherichia coli DNA photolyase to cis,syn dimers in four sequence contexts and to two cis,syn dimers in close proximity and on opposing strands. We found that the sequence within a 10-base pair region had little effect on binding and that two enzyme molecules bound to substrate containing two dimers in the 5'-staggered orientation, but not in the 3'-staggered one. Similarly, the excision of a cis,syn dimer by (A)BC excinuclease was not influenced by the sequence in the immediate vicinity of the dimer, and the enzyme was active on 5'-staggered cis,syn dimers, but not on 3'-staggered ones. Of special significance, we found that (A)BC excinuclease removed the cis,syn, trans,syn-I, (6-4), and Dewar photoproducts at vastly differing relative rates of 1:6:9:9, respectively.