DNA damage and repair provoked by ethidium azide (EA) photoaffinity labeling in mouse leukemia cells was studied by measuring sedimentation properties of nucleoids in neutral sucrose gradients, and it was found that the strand opening step was faster than that which followed damage of cells by ultraviolet (UV) light. The two insults were compared at levels of damage which gave the same overall rates of repair synthesis in intact cells and which required the same length of time to complete repair, as judged by the restoration of supercoiling of the isolated nucleoids. In the case of UV, single-strand breaks in DNA were detectable at 30 min, maximum at 2 h, and the superhelical properties restored at 21 h. With photoaffinity labeling, single-strand breaks were prominent immediately, even when photolabeling of cells was done on ice, but restoration of DNA supercoiling still required 21 h. Photolabeling of isolated nucleoids or isolated viral DNA with EA failed to introduce DNA strand breaks. However, it was discovered that photoaffinity labeling of DNA with EA resulted in alkali labile sites shown by single strand breaks produced on alkaline sucrose sedimentation or by alkali exposure followed by sedimentation on neutral formamide gradients. These results suggest that the drug attachment sites should be identifiable by the location of such single strand breaks.