Strand breaks in hamster lung DNA were analyzed following in vivo exposure to bleomycin. An alkaline unwinding procedure involving separation of single-strand from double-strand DNA by hydroxylapatite chromatography followed by fluorescence detection of DNA with bisbenzimide was adapted for these studies. Procedures were developed that allowed preparation of DNA from lungs of control animals with a relatively low amount of single-strand DNA relative to double-strand DNA. Time dependence of unwinding was demonstrated using samples that were damaged deliberately by brief probe sonication. To verify that residual bleomycin remaining in lungs at the time of sacrifice did not cause strand breaks during sample preparation, bleomycin was added to minced lung in vitro. Under these conditions, the ratio of single-strand to double-strand DNA was not increased significantly. Substantial strand breaks were produced in vivo at 15 min and 1 h following intratracheal instillation of bleomycin into hamsters, as evidenced by a 5-6-fold increase in the ratio of single-strand to double-strand DNA relative to controls. The DNA damage appeared to be repaired within 1 day following exposure.