Transcription through the late simian virus 40 (SV40) sequences have been examined in vivo and in vitro for identifying attenuation signals involved in regulating late RNA transcription. In addition to the previously identified and characterized attenuator 1 situated 93 nucleotides downstream from the major late transcription start site, a second attenuator, attenuator 2, situated 55 nucleotides downstream from it, has been identified. Attenuated transcripts mapping to this site have been observed in vivo as well as in several different in vitro transcription systems. The signal inducing transcription attenuation has been localized to a 21-base pair DNA fragment and has been found to function independently of the promoter directing transcription or its distance from the transcription start site. Furthermore, this attenuator, unlike that preceding it, does not include a region of dyad symmetry or A+T-rich sequences. Also, similar to the adenovirus 2 attenuator, but in contrast to SV40 attenuator 1, the block to elongation at the SV40 attenuator 2 appears to be modulated by the general transcription factors. It is concluded that in SV40 there are at least two types of attenuators: one that is dependent on RNA secondary structure and a second that is sequence specific and is modulated, at least in part, by the general transcription factors.