The synergistic action of hepatocyte nuclear factor (HNF)-1 and HNF-4 plays an important role in expression of the ₁-antitrypsin (₁-AT) gene in human hepatic and intestinal epithelial cells. Recent studies have indicated that the ₁-AT gene is also expressed in human pulmonary alveolar epithelial cells, a potentially important local site of the lung antiprotease defense. In this study, we examined the possibility that ₁-AT gene expression in a human pulmonary epithelial cell line H441 was also directed by the synergistic action of HNF-1 and HNF-4 and/or by the action of HNF-3, which has been shown to play a dominant role in gene expression in H441 cells. The results show that ₁-AT gene expression in H441 cells is predominantly driven by HNF-1, even though HNF-1 has no effect on ₁-AT gene expression in human hepatic Hep G2 and human intestinal epithelial Caco-2 cell lines. Expression of ₁-AT and HNF-1 was also demonstrated in primary cultures of human respiratory epithelial cells. HNF-4 has no effect on ₁-AT gene expression in H441 cells, even when it is cotransfected with HNF-1 or HNF-1. HNF-3 by itself has little effect on ₁-AT gene expression in H441, Hep G2, or Caco-2 cells but tends to have an upregulating effect when cotransfected with HNF-1 in Hep G2 and Caco-2 cells. These results indicate the unique involvement of HNF-1 in ₁-AT gene expression in a cell line and primary cultures derived from human respiratory epithelium.