Transcription activation by the Escherichia coli cyclic AMP receptor protein (CRP) at different promoters has been studied using RNA polymerase holoenzyme derivatives containing two full-length alpha subunits, or containing one full-length alpha subunit and one truncated alpha subunit lacking the alpha C-terminal domain (alpha CTD). At a promoter having a single DNA site for CRP, activation requires only one full-length alpha subunit. Likewise, at a promoter having a single DNA site for CRP and one adjacent UP-element subsite (high-affinity DNA site for alpha CTD), activation requires only one full-length alpha subunit. In contrast, at promoters having two DNA sites for CRP, or one DNA site for CRP and two UP-element subsites, activation requires two full-length alpha subunits. We conclude that a single copy of alpha CTD is sufficient to interact with one CRP molecule and one adjacent UP-element subsite, but two copies of alpha CTD are required to interact with two CRP molecules or with one CRP molecule and two UP-element subsites.