Simian virus 40 (SV40) is an excellent model system for investigating the cis- and trans-acting factors involved in eukaryotic DNA replication because it uses host enzymes, with the exception of the virus-encoded T-antigen (T-ag), to replicate its genome. Although its origin of replication (ori) is essential for DNA replication, there are transcriptional promoters and enhancers that affect DNA replication efficiency. T-ag binds to sites I to III within and around ori with different affinities and induces structural changes. We were interested in determining if the position of the promoters relative to ori influences the binding of T-ag to these regions. Furthermore, we characterized the DNA structural changes that occur as a result of protein binding when the promoters are absent and also when the promoters are moved from their wild-type position upstream of ori to a position downstream of ori. Using sequence- and conformation-specific chemical probes, our data indicate that (i) the conformation of site I is influenced by T-ag binding and by flanking sequences, (ii) the conformation of the promoters after T-ag binding is dependent on their location, and (iii) unwinding of ori is influenced by the location of the promoters and their presence or absence. These differences in DNA conformation may help explain decreases in relative DNA replication efficiency that occur when the promoters are absent or located downstream of ori.