To map the polyomavirus large T antigen binding sites on the viral genome we employed a quantitative immunoassay. Defined, radiolabeled fragments of the viral genome were reacted with crude nuclear extracts prepared from lytically infected mouse 3T6 cells, and the fragments bound by large T antigen were immunoprecipitated with anti-T serum and Formalin-fixed Staphylococcus aureus. The immunoprecipitated DNA was then analyzed by gel electrophoresis and autoradiography. By the use of a variety of restriction endonuclease-generated fragments of wild-type and mutant viral DNAs, the region of high-affinity binding was localized to a 153-base-pair stretch between nucleotides 5292 and 152. At least two independent binding sites lie within this region, one upstream and the other downstream of the Bg/I site at nucleotide 87. One of the binding sites is located within sequences required in cis for DNA replication; the other overlaps the TATA box and cap sites of the early transcription unit. The two sites share a common sequence, A/TGAGGC-N4/5-A/TGAGGC, which may serve as the recognition sequence for large T antigen.