Exposure of mouse kidney cells to 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB) (75 to 300 microM) during the late phase of infection by polyoma virus resulted in nearly complete (90 to 98%) inhibition of virus RNA synthesis. Sedimentation analysis revealed that, although the synthesis of high mol. wt. (greater than 10S) virus RNA was inhibited in a manner parallel to that of total virus RNA, the synthesis of small (3S to 7S) virus RNA was inhibited by only 40 to 50% in the presence of DRB. As a result, virus RNA synthesized in the presence of DRB contained a peak at 3S to 7S in addition ot residual high mol. wt. virus RNA. Small virus RNA from either untreated or DRB-treated cells contained three- to sixfold higher levels of transcripts from the DNA fragment which lies between the BamHI and Bg/I sites (58 . 0 to 72 . 2 map units) than from DNA fragments covering the rest of the virus genome. Furthermore, 80% of the small RNA which hybridized to this fragment was complementary to the L strand of virus DNA. These results suggest that L strand transcripts are initiated within the Bg/I-BamHI DNA fragment and that a portion of these transcripts is prematurely terminated within several hundred nucleotides of the site(s) of initiation. DRB had little effect on the synthesis of these prematurely terminated RNAs.