The aim of this study was to investigate aspects of transcription of highly expressed yeast genes. To investigate the sequence requirements for transcriptional initiation putative promoters were constructed which contained promoter elements subcloned from the 5'-flanking region of the phosphoglycerate kinase gene (PGK1). The analysis of these promoters was hindered by high levels of transcriptional readthrough from adjacent sequences in the promoter probe vector. A CT-rich block, found upstream of many highly expressed yeast genes has previously been shown to influence the position of the transcriptional initiation site (I-site). Preliminary data presented here is consistent with the existence of a yeast protein which specifically interacts with this region.