Members of the forkhead/winged-helix family of transcription factors are expressed in tissue-specific patterns and play critical roles in development and cell differentiation. The expression of forkhead family member hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) has been localized by RNA-blot analysis and in situ hybridization to the proximal airway of the lung (trachea, bronchi, and bronchioles) with onset at mouse embryonic day (E) 14.5 and is present in the choroid plexus, ependymal cells, oviduct, and testis. We hypothesized that the restricted expression of HFH-4 messenger RNA suggests a function common to these tissues and therefore a cell-specific role for HFH-4. Accordingly, an anti-HFH-4 antibody was generated and used for cell-specific localization of protein expression to begin to identify the functions of HFH-4. We found HFH-4 expression in proximal airway ciliated epithelial cells, but not Clara cells or alveolar epithelial cells. HFH-4 was also expressed in ciliated epithelial cells of the nose and paranasal sinuses, choroid plexus, ependyma, and oviduct. In developing mouse lung, HFH-4 expression was initially detected in airway epithelial cells at E15.5, before the appearance of cilia, and at later stages was localized to epithelial cells with cilia. In the testis, HFH-4 expression in spermatids was coincident with stage-specific generation of flagella. The temporal relationship of HFH-4 expression to the development of cilia and flagella, and the restricted expression in ciliated epithelial cells, suggest that this transcription factor has a role in regulation and maintenance of the ciliated cell phenotype in epithelial cells.