Thyroid transcription factor 1 (TTF-1), hepatocyte nuclear factor 3alpha (HNF-3alpha), and HNF-3beta regulate the transcription of genes expressed in the respiratory epithelium. To test whether members of the HNF-3/forkhead family influence TTF-1 gene expression, deletion constructs containing the 5' region of the human TTF-1 gene were transfected into immortalized mouse lung epithelial (MLE) cells. DNase I protection and electrophoretic mobility shift assays identified elements in the 5' region of the TTF-1 gene that bound MLE cell nuclear proteins consistent with the binding of HNF-3 to sites at positions -135 to -124 and -14 to -3. In MLE cells, TTF-1-luciferase reporter constructs were activated by cotransfection with HNF-3beta, activated to a lesser extent by HNF-3alpha, but not activated by HFH-8. HNF-3alpha. and HFH-8 inhibited the activation of TTF-1-luciferase by HNF-3beta. Site-specific mutagenesis of each of the HNF-3 binding sites in the human TTF-1 gene inhibited the binding of MLE cell nuclear proteins and inhibited transactivation of the TTF-1-luciferase constructs after cotransfection with HNF-3beta. Immunohistochemical staining demonstrated that both HNF-3beta and TTF-1 were detected in bronchiolar and alveolar type II cells in the human lung. Modulation of TTF-1 gene expression by members of the HNF-3/forkhead family members may provide a mechanism by which distinct HNF-3/forkhead family members influence respiratory epithelial cell gene expression and cell differentiation.