In vivo the poly(A) signal not only directs 3'-end processing but also controls the rate and extent of transcription. Thus, upon crossing the poly(A) signal RNA polymerase II first pauses and then terminates. We show that the G/U-rich region of the poly(A) signal, although required for termination in vivo, is not required for poly(A)-dependent pausing either in vivo or in vitro. Consistent with this, neither CstF, which recognizes the G/U-rich element, nor the polymerase CTD, which binds CstF, is required for pausing. The only part of the poly(A) signal required to direct the polymerase to pause is the AAUAAA hexamer. The effect of the hexamer on the polymerase is long lasting--in many situations polymerases over 1 kb downstream of the hexamer continue to exhibit delayed progress down the template in vivo. The hexamer is the first part of the poly(A) signal to emerge from the polymerase and may play a role independent of the rest of the poly(A) signal in paving the way for subsequent events such as 3'-end processing and termination of transcription.