When readily used nitrogen sources are available, the expression of genes encoding proteins needed to transport and metabolize poorly used nitrogen sources is repressed to low levels; this physiological response has been designated nitrogen catabolite repression (NCR). The cis-acting upstream activation sequence (UAS) element UAS(NTR) mediates Gln3p-dependent, NCR-sensitive transcription and consists of two separated dodecanucleotides, each containing the core sequence GATAA. Gln3p, produced in Escherichia coli and hence free of all other yeast proteins, specifically binds to wild-type UAS(NTR) sequences and DNA fragments derived from a variety of NCR-sensitive promoters (GDH2, CAR11 DAL3, PUT1, UGA4, and GLN1). A LexA-Gln3 fusion protein supported transcriptional activation when bound to one or more LexAp binding sites upstream of a minimal CYC1-derived promoter devoid of UAS elements. LexAp-Gln3p activation of transcription was largely independent of the nitrogen source used for growth. These data argue that Gln3p is capable of direct UAS(NTR) binding and participates in transcriptional activation of NCR-sensitive genes.