In vitro transcription analyses were used to establish the biological function of a 43-kDa affinity-purified DNA-binding protein. The 43-kDa affinity-purified protein protects the region from position -59 to position -35 of the somatostatin promoter from DNase I digestion. This region of the somatostatin promoter harbors the TGACGTCA motif, also found and required for function in a number of other cAMP-responsive and adenovirus E1A-inducible promoters. Efficient and authentic transcription in vitro directed from the somatostatin promoter requires the TGACGTCA promoter element. In vitro transcription assays performed in the presence of somatostatin (positions -60 to -29), enkephalin (positions -105 to -71), and adenovirus type 5 E3 gene (positions -72 to -42) competitor fragments, harboring similar TGACGTCA motifs, selectively inhibit transcription directed from the somatostatin promoter, suggesting that the TGACGTCA element is the site of interaction of a somatostatin gene transactivator. Furthermore, extracts depleted of the TGACGTCA-binding activities by affinity chromatography utilizing a biotinylated oligonucleotide-avidin resin, are incapable of directing transcription from the somatostatin but not from the adenovirus major late promoter. Addition of the purified 43-kDa protein to the affinity-depleted extract restores transcription from the somatostatin promoter. These results are consistent with the 43-kDa protein being a trans-activator of the somatostatin gene.