Proteins bound to SV 40 DNA in sarkosyl-treated nuclei have been studied. The major component is topoisomerase I, a 60-70 kDa protein which possesses a strong DNA-nicking activity in the presence of sarkosyl and camptothecin. An SV 40 DNA fraction containing sarkosyl-resistant proteins constitutes 2 to 3% of the total nuclear SV 40 DNA and is enriched in transcriptionally active DNA (as monitored by distribution of RNase-resistant in vivo pulse-labeled RNA). An SV 40 DNA fraction, which is nicked due to covalent binding of topoisomerase I upon sarkosyl treatment is also enriched in transcriptionally active DNA. Topoisomerase I cleavage sites on SV 40 DNA which arise following sarkosyl treatment of nuclei or camptothecin treatment of infected cells have been mapped. The strongest site is located at nucleotide 381 on the non-coding strand and is framed by nuclease hypersensitive sites.