The growth hormone (GH) gene is specifically expressed in the anterior pituitary. The first 289 bp of the human (h)GH 5' region contains a promoter that is active only in pituitary-derived cell lines. These cell lines also contain a specific factor, GHF-1, that binds to two sites upstream to the hGH TATA box. Using in vitro transcription systems we demonstrate greater hGH promoter activity in extracts of GH-expressing GC cells than in extracts of nonexpressing HeLa cells. The two GHF-1 binding sites are essential promoter elements in GC extracts. Addition of GHF-1-containing fractions to HeLa extracts stimulates hGH promoter activity. Thus, GHF-1 appears to be a positively acting, cell-type- and promoter-specific transcription factor. Furthermore, the tissue specificity of the hGH promoter appears to be positively controlled by the abundance or activity of GHF-1.