NIT2, a global positive-acting regulatory protein in Neurospora crassa, activates the expression of a series of unlinked structural genes in the nitrogen regulation circuit. NIT2 binding sites in the promoter region of the nit-3, alc, and lao genes are very different in sequence context except for the presence of at least two copies of a GATA core sequence. Changing a single nucleotide of only one of two closely spaced GATA core elements abolished NIT2 binding, demonstrating their importance for NIT2 binding. The effect of altering the number, orientation, or spacing of paired GATA elements and the importance of 5'- and 3'-flanking sequences upon NIT2 binding were examined. Strong binding sites for a NIT2-beta GAL fusion protein appear to contain at least two GATA elements, which can have varied spacing but must be within a certain effective distance, approximately 30 bp, of each other. Surprisingly, the orientation of GATA elements and their flanking sequences have only modest effects upon NIT2 binding.