We determined the ability of the 40 000-dalton Escherichia coli photoreactivating enzyme to act on a variety of pyrimidine-pyrimidine photoproduct substrates in nucleic acids. The enzyme is at least as active on cis-syn-cyclobutylpyrimidine dimers in supercoiled DNA as in linear DNA, but inactive on dimers in RNA. Both the phosphodiester bond internal to the deoxyriboses of the pyrimidines of the dimer and the N-glycosyl bond joining the pyrimidine to deoxyribose must be intact for enzyme action. The enzyme has no activity toward (6-4) pyrimidine-cytosine products in DNA.