Single and double strand breakage of DNA by bleomycin under various conditions was analyzed quantitatively using purified simian virus 40 (SV40) DNA and SV40 chromatin. The method employed for DNA assay was fluorometry of SV40 DNA separated into three forms I, II, and III by 1.4% agarose gel electrophoresis and stained with ethidium bromide. This method was extremely simple and accurate for quantitative estimation of single and double strand breakage of circular DNA. The amount of bleomycin required for in vitro breakage of SV40 chromatin DNA was much higher than that of naked SV40 DNA. 2-Mercaptoethanol accerated bleomycin-induced DNA strand breakage. The concentration of 2-mercaptoethanol required for the acceleration was higher in SV40 chromatin than in SV40 DNA. DNA strand breakage was induced by ferrous ions in the absence of bleomycin to a similar level in SV40 chromatin and in SV40 DNA. The acceleration of bleomycin-induced strand breakage by a low concentration of ferrous ion was higher in SV40 DNA than in SV40 chromatin. Ethylenediaminetetraacetic acid (EDTA) and copper ions were not efficient for stopping the reaction of bleomycin-induced DNA breakage. Sodium dodecyl sulfate(SDS) was a useful reagent for stopping the reaction.