The control of gene transcription is usually mediated by transacting transcriptional factors that bind to upstream regulatory elements. As insulin regulates transcription of the growth hormone (GH) gene, we tested nuclear extracts from unstimulated and insulin-stimulated Chinese hamster ovarian (CHO) cells for binding to four human GH (hGH) gene promoter oligonucleotide fragments identified as target-binding sequences by DNAse I footprinting. Using a mobility shift assay, an insulin-induced DNA-binding protein was identified. This protein binds to two upstream overlapping oligonucleotide sequences. Binding activity is present at low levels in unstimulated CHO cells and is stimulated by insulin treatment with a time course suggesting that protein synthesis is required. Incubation of the cells with cycloheximide and puromycin confirmed that de novo protein synthesis is necessary for the increased binding activity. Competition with excess unlabeled specific competitor oligonucleotides prevented binding, while unrelated similar-sized oligonucleotides failed to compete for binding, indicating that the observed DNA-protein complex formation is specific. A protein of approximately 70-80 kD was detected by gradient gel electrophoresis. In conclusion, insulin-mediated DNA-protein binding has been identified on the upstream hGH promoter, suggesting a trans-active role for insulin in mediating polypeptide hormone gene expression.