Constitutively expressed Fas ligand in several distinct epithelial cell types appears to protect tissues by inducing apoptosis of Fas(+) immune cells during inflammatory reactions. To study the transcriptional regulation of Fas ligand gene in airway epithelial cells, a 618-bp 5'-flanking region of mouse Fas ligand gene was cloned, sequenced, and the transcriptional start site was determined by using 5'-RACE. Deletion analysis, gel mobility shift assays and site-directed mutagenesis indicated that a CCAAT box located -214 bp upstream from the transcription start site served as a major positive regulatory cis-element in an airway epithelial cell line. This element was not required for constitutive Fas ligand expression in Sertoli cells. Furthermore, the activity of the site did not involve the NF-Y protein complex or c/EBP protein family. UV-cross linking proteins to this element indicated that a approximately 23-kDa transcription factor bound to the Fas ligand promoter CCAAT box and, thus, likely plays an important role in the regulation of Fas ligand expression in airway epithelial cells.