Expression of the human-growth-hormone gene is restricted to pituitary somatotrophs. Two protein-DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary-specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease-I protection and gel-retardation experiments. The first footprint, located between -257 and -290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides -257 and -267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides -263 and -290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I. The second footprint, located between nucleotides -116 and -140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human-growth-hormone gene is therefore a candidate for regulation by these factors in vivo.