Virus-specific RNA synthesized in monkey cells after infection by both wild-type simian virus 40 (SV40) and the early SV40 temperature-sensitive mutant tsA58 has been analyzed. The fraction of SV40-specific RNA increased throughout infection with either wild-type SV40 or with tsA58 in direct proportion to the accumulation of progeny DNA molecules, suggesting their role in the late transcriptional process. Cytoplasmic fractions from cells infected at various temperatures (31.5 to 41 degrees C) by wild-type virus and harvested 48 h later contained 4 to 8% virus-specific RNA, of which 5 to 10% was early SV40 RNA. In contrast, though 5 to 8% of the cytoplasmic RNA from tsA 58-infected cells incubated at 31.5 to 37 degrees C for 48 h was virus specific, the percentage of early virus-specific RNA ranged from 25 to 80% as the incubation temperature increased. In tsA58-infected cultures incubated for 48 h at 41 degrees C (a temperature at which essentially no tsA 58 DNA synthesis occurred), only 0.4% of the cytoplasmic RNA was virus specific, but at least 90% of this RNA was early. In experiments where cells were inoculated at 32 degrees C and shifted at 48 h postinfection to 40 degrees C for various times, the percentage of virus-specific pulse-labeled RNA varied from 3.5 to 10.0%. Of the virus-specific RNA, early SV40 RNA ranged from 14 to 65% in tsA 58-infected cultures. Analogous studies with Sarkosyl-extracted viral transcription complexes to incorporate label into nascent (unprocessed) viral RNA yielded essentially identical results. This finding strongly suggests that the overproduction of early SV40 RNA occurs at the level of synthesis. While cytosine arabinoside effectively terminated most viral DNA replication in wild-type-infected cells, the ratio of early to late viral RNA remained less than 1:9. These results demonstrate that: (1) the amount of virus-specific RNA synthesized depends directly on the amount of viral DNA available for use as templates; once viral DNA replication has occurred, presumably providing progeny SV40 DNA molecules for templates, the level of transcription remains high; (ii) termination of viral DNA replication does not terminate late SV40 transcription; (iii) early SV40 RNA is overproduced by tsA 58 at all temperatures, but especially at higher temperatures; and (iv) overproduction of early SV40 RNA appears to be correlated with defectiveness of the tsA mutant T-antigen. These results suggest that T-antigen may regulate its own production either by repressing the synthesis of early viral RNA or by stimulating the synthesis of late SV40 RNA or both.