General regulatory factor I (GRFI) is a yeast protein that binds in vitro to specific DNA sequences at diverse genetic elements. A strategy was pursued to test whether GRFI functions in vivo at the sequences bound by the factor in vitro. Matches to a consensus sequence for GRFI binding were found in a variety of locations: upstream activating sequences (UASs), silencers, telomeres, and transcribed regions. All occurrences of the consensus sequence bound both crude and purified GRFI in vitro. All binding sites for GRFI, regardless of origin, provided UAS function in test plasmids. Also, GRFI binding sites specifically stimulated transcription in a yeast in vitro system, indicating that GRFI can function as a positive transcription factor. The stimulatory effect of GRFI binding sites at UASs for the PYK1 and ENO1 genes is significantly enhanced by flanking DNA elements. By contrast, regulatory sequences that flank the GRFI binding site at HMR E convert this region to a transcriptional silencer.