Heteroduplexes were formed between SV40 replication origin-containing DNA fragments derived from wild-type genomic DNA and a viral deletion mutant (d1295) lacking 18 base pairs from a 27 bp inverted repeat that is imbedded within the minimal replication origin sequence. Among these structures were molecules bearing a 7 bp stem and a 3 nucleotide loop on the wild-type strand between nucleotides 5230 and 6. By nondenaturing gel electrophoresis, heteroduplex molecules bearing such a structure on the E-strand could be separated from those with the complementary structure on the L-strand, and either heteroduplex could be so separated from either the wild-type or d1295 homoduplex fragment. Each of these forms was singly 5′ or 3′ end-labeled and then evaluated as a target for specific binding to purified SV40 large T antigen isolated from wild-type virus-infected cells. The results of DNAase footprint protection analyses showed that the existence of both the E- and L-strand stem-loop structures inhibited T binding to site 2 which composes much of the minimal origin sequence. By contrast, T bound readily to this site in both homoduplexes. Furthermore, T protected both E- and L-strand sequences of its strongest binding site (site 1)—which abuts the early side of site 2—when the site 2 stem-loop was on the L-strand, and protected L-strand site 1 sequences normally when the stem-loop was on the E-strand. However, a marked alteration of binding to site 1 E-strand sequences was noted when the stem-loop was on the E-strand. Thus this alteration in replication origin secondary structure resulted in discrete local and vicinal effects on T binding. Furthermore, the results suggest that within site 1—a sequence employed as an early transcriptional regulatory locus—T antigen can bind specifically and tightly to one strand without exhibiting similar behavior on the other strand.