Complexes between simian virus 40 DNA and topoisomerase I (topo I) were isolated from infected cells treated with camptothecin. The topo I break sites were precisely mapped by primer extension from defined oligonucleotides. Of the 56 sites, 40 conform to the in vitro consensus sequence previously determined for topo I. The remaining 16 sites have an unknown origin and were detectable even in the absence of camptothecin. Only 11% of the potential break sites were actually broken in vivo. In the regions mapped, the pattern of break sites was asymmetric. Most notable are the clustering of sites near the terminus for DNA replication and the confinement of sites to the strand that is the template for discontinuous DNA synthesis. These asymmetries could reflect the role of topo I in simian virus 40 DNA replication and suggest that topo I action is coordinated spatially with that of the replication complex.Topoisomerases are enzymes that change the superhelical state of DNA by transiently breaking one or both strands of the duplex. Type II topoisomerases make a staggered double-strand break, whereas the type I enzymes introduce a single-strand break. The known eucaryotic topoisomerases are capable only of relaxing supercoiled DNA, whereas some of the bacterial enzymes are able to introduce supercoils (e.g., DNA gyrase in Escherichia coli) (for recent reviews, see references 39 and 62 to 64). The eucaryotic type