Levels of transcription within the E and L strands of the five major PstI fragments of polyomavirus (strain AT3) were measured by pulse-labeling RNA both in infected cells and in isolated nuclei or viral transcription complexes during the late phase of infection. Quantification was assured by hybridization to single-stranded DNAs in solution followed by collection of hybrids on nitrocellulose filters and ribonuclease treatment. The level of in vivo transcription in the region of the early (E strand) promoter was two- to threefold higher than that in all other E-strand regions, suggesting that most RNA polymerases prematurely terminate transcription shortly downstream from this promoter during the late phase. In vitro transcription levels in this region were five- to tenfold higher than in the remainder of the E strand, suggesting that many RNA polymerases 'stall' shortly after initiation in vivo but can be reactivated and continue transcription in vitro upon exposure to detergents and high salt solution. Some premature termination nearby the late (L strand) promoter was also detected by the same method. Strikingly, many RNA polymerases also stalled on the L strand in the region of the early promoter, some 5 x 10(3) bases downstream from the late promoter. Treatment of cells with dichlororibofuranosylbenzimidazole did not affect polymerases that stalled or terminated prematurely, but strongly reduced the presence of polymerases that normally transcribed throughout the entire E or L strand. Examination of the size of RNA chains produced during in vitro incubations showed that many polymerases stalled in vivo within 50 to 100 nucleotides downstream from the initiation sites on both DNA strands. The number of polymerases active in vitro at the E strand promoter was similar to the number of polymerases at the L strand promoter. However, in contrast to L-strand transcription, most of the polymerases that initiated at the E-strand promoter were incapable of extended transcription in vivo. These results suggest that large T antigen-mediated repression of E-strand transcription is not simply due to the exclusion of RNA polymerases from the early promoter. Stalling and/or premature termination by RNA polymerases shortly downstream from the early promoter appears to be a mechanism by which temporal regulation of polyomavirus gene expression can be effected.