Forkhead transcription factors appear to play important roles in cell fate determination and cell-specific gene expression during development. To study the role of these transcription factors in human development and disease, a human forkhead transcription factor HFH-4 was isolated by screening a human fetal lung cDNA library with a rat HFH-4 probe. Sequence analysis of isolated clones revealed an open reading frame of 421 amino acids with a conserved 100 amino acid DNA binding domain. Comparison of the human amino acid sequence to the murine and rat proteins revealed greater than 90% amino acid identity overall and 100% identity within the DNA binding domain. Comparison to previously identified human forkhead DNA binding domains revealed less than 50% amino acid homology. The human cDNA is encoded by a single Hfh-4 gene localized to chromosome 17q21-25 by Southern blot analysis of human/hamster somatic cell hybrid DNA. Examination of adult human tissues by RNA blot analysis revealed expression of a 2.5 kb HFH-4 transcript in male and female reproductive tissues, lung, and brain. In human fetal tissue at 22 to 24 weeks gestation a 2.5 kb transcript was present in lung, kidney and brain. By in situ hybridization, expression of HFH-4 in the human fetal lung was restricted to the developing bronchiolar epithelium. Expression of HFH-4 in developing human organs suggests a role for this transcription factor in regulating human developmental processes.