The sequential actions of two enzymes believed to be involved in DNA repair, namely a mammalian endonuclease and the bacterial DNA polymerase I on psoralen bound 32P-labeled DNA, was studied. When ultraviolet-irradiated DNA is exposed to the sequential action of the endonuclease, the formation of single-strand breaks prepares the DNA for the exonucleolytic excision of thymine dimers. The mammalian endonuclease purified from rat liver to electrophoretic homogeneity is inactive on normal DNA, DNA irradiated at 360 nm or DNA mixed with psoralen without irradiation. Incubation of psoralen-bound DNA labeled with 32P with the endonuclease releases the isotope in the acid soluble indicating that psoralen-bound DNA is susceptible to the endonucleolytic attack. Sedimentation of DNA on sucrose gradients indicates that there is no collapse of the DNA molecule after treatment with the endonuclease. Moreover, there is no release of the adduct in the acid soluble after treatment with DNA polymerase, indicating that the 5--3 min exonucleolytic activity of that enzyme is impaired by the remaining crosslinks. The crosslinks also inhibit the incorporation of [3H] dATP in presence of DNA polymerase I.