The Escherichia coli Crl protein has been described as a transcriptional coactivator for the stationary-phase sigma factor sigma(S). In a transcription system with highly purified components, we demonstrate that Crl affects transcription not only by the Esigma(S) RNA polymerase holoenzyme but also by Esigma(70) and Esigma(32). Crl increased transcription dramatically but only when the sigma concentration was low and when Crl was added to sigma prior to assembly with the core enzyme. Our results suggest that Crl facilitates holoenzyme formation, the first positive regulator identified with this mechanism of action.