The large T antigens of polyomavirus and simian virus 40 (SV40) recognize and bind to specific, noncoding DNA sequences which are located between the beginning of the early and late transcription units in their respective genomes. Each large T antigen binds to multiple sites within this intergenic DNA stretch. Polyomavirus large T antigen binds to at least two sites within its DNA, and SV40 large T antigen binds to three sites within SV40 DNA. Comparison of the DNA sequences which comprise the binding sites in polyomavirus DNA or those which make up the binding sites in SV40 DNA has led to recognition of a common sequence, -GAGGC-, which is repeated within each large-T-antigen-binding site. We tested the hypothesis that repeats of this pentanucleotide form the recognition-binding site for polyomavirus and SV40 large T antigen. This was accomplished by measuring the binding of each large T antigen to both polyomavirus and SV40 DNA and to synthetic DNA substrates which did or did not contain repeats of the -GAGGC- sequence. Polyomavirus large T antigen bound to specific fragments of SV40 DNA, and SV40 large T antigen bound with specificity to polyomavirus DNA. In each case, the DNA fragments bound by the heterologous large T antigen were the same as those bound by the homologous large T antigen. Moreover, polyomavirus and SV40 large T antigen only bound to synthetic DNA substrates which contained repeats of the pentameric sequence. This synthetic DNA also competed effectively with native polyomavirus or SV40 DNA as a substrate in binding reactions with one or the other large T antigen. These results led us to conclude that repeats of the -GAGGC- sequence form the recognition-binding site for both polyomavirus and SV40 large T antigen.