During transcription of the late strand of polyomavirus DNA, RNA polymerase II stalls and accumulates nearby the binding sites on viral DNA recognized by polyomavirus large T antigen. Stalling by RNA polymerases is eliminated when thermolabile large T antigen is inactivated by using a temperature-sensitive virus mutant (J. Bertin, N.-A. Sunstrom, P. Jain, and N. H. Acheson, Virology 189:715-724, 1992). To determine whether stalling by RNA polymerases is mediated through the interaction of large T antigen with one or more of its binding sites, viable polyomavirus mutants that contain altered large-T-antigen-binding sites were constructed. Point mutations were introduced by site-directed mutagenesis into the multiple, clustered G(A/G)GGC pentanucleotides known to be the target sequence for large T-antigen binding. Mutation of the G(A/G)GGC pentanucleotides in the first two binding sites encountered by RNA polymerases in the intergenic region (sites C and B) had no detectable effect on stalling as measured by transcriptional run-on analysis. However, mutation of the two GAGGC pentanucleotides in binding site A, which lies adjacent to the origin of viral DNA replication, eliminated stalling by RNA polymerases. We conclude that binding of large T antigen to site A blocks elongation by RNA polymerase II. Further characterization of virus containing mutated site A did not reveal any effects on early transcription levels or on virus DNA replication. However, the mutant virus gave rise to small plaques, suggesting impairment in some stage of virus growth. Stalling of RNA polymerases by large T antigen bound to the intergenic region of viral DNA may function to prevent transcription from displacing proteins whose binding is required for the normal growth of polyomavirus.