Lymphocyte cells (P-288) in culture were exposed to daunorubicin (Dm) and analyzed for DNA damage by means of alkaline sucrose and neutral (formamide) sucrose gradients. In neutral gradients, the sedimentation rate of DNA from cells treated with 25 X 10(-8) M Dm for brief periods up to 60 min is the same as that of the DNA from control cells (i.e., drug-treated for "zero" time). In contrast, extensive DNA damage is already apparent in alkaline gradients after treatment of the cells for only 30 min. After treatment for 2 hr, single-strand damage is evident in both types of gradient. These results demonstrate the introduction of alkali-labile regions in the DNA of cells treated for 45 or 60 min with Dm. The DNA damage is concurrent with inhibition of thymidine incorporation, and both may occur at threshold levels without affecting subsequent growth of the P-288 cells after short exposures to low concentrations of the drug.