Eukaryotic type 1 DNA topoisomerases break single-stranded DNA at specific sites. A preferred site for rat liver topoisomerase breakage in single-stranded phi X174 DNA was located within a region of the DNA with the potential for duplex formation. To investigate the relationship between sites of breakage in duplex and single-stranded DNA, a restriction fragment containing sequences from the transcriptional regulatory and enhancer region of the simian virus 40 genome was used as a substrate for topoisomerase. While different patterns of breakage in the native and denatured forms of the DNA were observed, some sites of breakage were common to both forms. The break sites in the denatured DNA were a subset of the break sites in the duplex DNA and were located in regions which had the potential for intrastrand base-pairing due to distal complementary sequences. A series of single-stranded fragments were generated with the distal complementary sequences deleted and these fragments were used as substrates for topoisomerase breakage. The lack of detectable breakage at a site when the complementary sequence was deleted, suggests that topoisomerase acts at duplex regions in the single-stranded DNA and that it is not active on regions of single-stranded DNA that are not base-paired.