All ribosomal protein (rp) gene promoters from Sac-charomyces cerevlslae studied so far contain either (usually two) binding sites for the global gene regulator Rap1p or one binding site for another global factor, Abf1p. Previous analysis of the rpS33 and rpL45 gene promoters suggested that apart from the Abf1 binding slte, additional cls-acting elements play a part in transcription activation of these genes. We designed a promoter reconstruction system based on the β-glucu-ronidase reporter gene to examine the role of the Abf1 binding site and other putative cls-acting elements In promoting transcription. An Isolated Abfl binding site turned out to be a weak activating element. A T-rich sequence derived from the rpS33 proximal promoter was found to be stronger, but full transcription activation was only achieved by a combination of these elements. Both in the natural rpL45 promoter and in the reconstituted promoter, a Rap1 binding site could functionally replace the Abfl binding site. Characteristic rp gene nutritional control of transcription, evoked by a carbon source upshift or by nitrogen re-feeding to nitrogen starved cells, could only be mediated by the combined Abfl (or Rap1) binding site and T-rich element and not by the individual elements. These results demonstrate that Abf1p and Rap1p do not activate rp genes in a prototypical fashion, but rather may serve to potentiate transcription activation through the T-rich element. © 1995 Oxford University Press.