Rat (rGH) is expressed exclusively in cells from the anterior lobe of the pituitary gland. Using DNAsel footprinting assays, we have examined both pituitary and nonpituitary cell nuclear extracts for proteins which bind specifically to the rGH promoter and 5'-flanking region. In agreement with previous studies, we have located binding sites between -96 and -65, and between -148 and -118 for proteins which have been termed GC1 and GC2, respectively. The GC2 footprint is found using extracts from both pituitary and nonpituitary cells, but GC1 is observed only in pituitary cells. We have also located a binding site for an additional pituitary-specific protein upstream from the GC1 binding site, between -241 and -220. The footprint for this protein, which we call GC3, is found with pituitary extracts, but not with extracts from nine other nonpituitary cell types. Although this pattern of activity is similar to that of GC1, competition experiments with synthetic oligonucleotides show that the two proteins are distinct. Deletion of the GC3 binding site has only a small effect on rGH promoter activity in transiently transfected pituitary cells and fibroblasts.