DNA polymerases from prokaryotic, eukaryotic, and oncornaviral sources are irreversibly inactivated upon exposure to Rose Bengal in the presence of light (photooxidation conditions). Inactivation of these enzymes under dark (nonoxidative) conditions is totally reversible [Srivastava, S. K., & Modak, M. J. (1982) Biochemistry 21, 4633-4639]. The primary effect of photooxidation on the enzyme-Rose Bengal complex was found to be the loss of template-primer binding ability within 5 min of exposure to light. The presence of template-primer but not the substrate deoxynucleotides consistently provided partial protection against Rose Bengal mediated photooxidative inactivation. Preformed enzyme-template-primer complexes were not dissociated by Rose Bengal under these conditions. However, prolonged exposure (25-30 min) of this complex to light in the presence of Rose Bengal led to complete inactivation of catalytic activity without affecting the ability of enzyme to bind to template-primer. These results clearly indicate that oxidative inactivation of avian myeloblastosis virus reverse transcriptase in the presence of Rose Bengal involves a domain within the enzyme that contains the template-primer binding site as well as an additional site which is required for the expression of both the polymerization and nuclease activities of the reverse transcriptase.