We have examined the discrete species of simian virus 40 (SV40) RNA present very early in infection of monkey cells with wild-type virus, with mutant tsA58 virus, and with the corresponding DNAs to distinguish between two classes of models for control of late transcription: (i) positive control mediated by large-T antigen and (ii) negative control mediated by a repressor protein associated with viral DNA in the virion. Total cytoplasmic or nuclear polyadenylated RNAs from infected cells were denatured with glyoxal, separated by electrophoresis on agarose gels, and transferred to diazobenzyloxymethyl paper. The positions of specific early and late RNA species were determined with region-specific SV40 DNA probes. The technique can detect individual RNAs present at the level of less than one copy per cell. After 9.5 h at 37°C, appreciable amounts of two early RNAs (2.6 kilobases [kb] and 2.9 kb) were present in the cytoplasm of cells infected with wild-type virus or DNA, along with much smaller amounts of two late RNAs, 1.6 kb (16S) and 2.5 kb (19S). The amounts of the late RNAs were reduced, but they were still synthesized in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. In comparable infections with tsA58 virus or DNA at nonpermissive temperature (41°C), substantial amounts of the two early RNAs were again present, but the two late RNAs could not be detected. However, small amounts of the late RNAs were found when infections with tsA58 virus or DNA were prolonged to 30 h at 41°C. These results are not consistent with negative control of late transcription through the action of a repressor and, taken together with other data, suggest that T antigen has an active role in late RNA synthesis. Specific early and late viral RNAs were also detected in the nuclear poly(A)+ fractions and were similar in size to the RNA species found in the cytoplasmic polyadenylated fractions. The late nuclear RNAs (1.8 and 2.9 kb) were significantly larger than the late cytoplasmic species, possibly because they are precursors. The 2.6- and 2.9-kb early RNAs found in the cytoplasm are probably the messengers for large-T and small-t antigens, respectively.