Gene expression can be defined as the conversion of information existing in a molecule of DNA into a mature RNA or protein product and each step in the process, which requires the concerted action of several macromolecules for completion, may be perturbed by the post-translational modification of specific proteins with ADP-ribose. The participation of poly(ADP-ribose) in the regulation of transcription initiation was examined using cell-free systems for both ribosomal RNA and ribosomal proteins. The presence or absence of poly(ADP-ribose) polymerase did not influence the transcription process. Similarly, under conditions optimal for poly(ADP-ribose) polymerase activity, no change in transcription was observed. A direct contribution of poly(ADP-ribosyl)ation to gene transcription thus could not be detected. In contrast, the addition of 3-aminobenzamide to quiescent hepatoma cells treated with insulin inhibited the stimulation of rRNA synthesis. The high concentrations necessary for this effect suggest that a mono(ADP-ribosyl)ation event participates in the cellular action of insulin. A role in the signal transduction pathway leading to activation of rRNA gene expression has been proposed.