Analysis of the upstream activation sequence (UAS) of the yeast phosphoglycerate kinase gene (PGK) has demonstrated that a number of sequence elements are involved in its activity and two of these sequences are bound by the multifunctional factors Rap1p and Abf1p. In this report we show by in vivo footprinting that the regulatory factor encoded by GCR1 binds to two elements in the 3' half of the PGK UAS. These elements contain the sequence CTTCC, which was previously suggested to be important for the activity of the PGK UAS and has been shown to be able to bind Gcr1p in vitro. Furthermore, we find that Gcr1p positively influences PGK transcription, although it is not responsible for the carbon source dependent regulation of PGK mRNA synthesis. In order to mediate its transcriptional influence we find that Gcr1p requires the Rap1p binding site, in addition to its own, but not the Abf1p site. As neither a Rap1p nor a Gcr1p binding site alone is able to activate transcription, we propose that Gcr1p and Rap1p interact in an interdependent fashion to activate PGK transcription.