Epithelium-derived nitric oxide (NO) plays an important role in airway function. We have previously shown that the endothelial isoform of NO synthase(NOS), or eNOS, is constitutively expressed in airway epithelium, and that its pulmonary expression is limited solely to airway epithelium and endothelium. The purpose of the present study was to determine the molecular basis of cell-specific eNOS expression in the airway epithelium. The human eNOS promotor fused to a luciferase reporter gene was transfected into NCI-H441 human bronchiolar epithelial cells, which express eNOS constitutively. Transfection of H441 cells with 1624 bp of the promotor sequence 5' to the initiation ATG (-1624) yielded a 19-fold increase in promotor activity relative to transfection with vector alone. Similarly, -1624 yielded a 35-fold increase in promotor activity in pulmonary endothelial cells. However, there was no change in promotor activity with -1624 transfected into CCD-18Lu lung fibroblasts, consistent with cell-specific eNOS expression in airway epithelium and endothelium. There was 6-fold greater promotor activity in the H441 cells, which are of Clara cell origin, compared to the non-Clara cell human airway epithelial lines, BEAS-2B and NHBE. Progressive 5' deletion mutants of the eNOS promotor were then generated to determine the putative regulatory domains underlying cell-specific expression in airway epithelium. Deletion from -1624 to -994, -318, and -279 did not alter basal eNOS promotor activity. However, further deletion from -279 to -248 reduced basal promotor activity by 65%. Promotor activity was completely lost with deletion to -79. These findings suggested that positive regulatory elements reside between -279 and -248 and between -248 and -79. Point mutations of the -1624 promotor revealed that the positive regulatory element between -279 and -248 is the consensus GATA binding motif at -254, and that the positive regulatory element between -248 and -79 is the Sp1 binding motif at -125. We conclude that airway epithelial eNOS is preferentially expressed in Clara cells, and that its cell-specific expression in airway epithelium is mediated by GATA and Sp1 sites in the core eNOS promotor. Further studies of eNOS promotor function in epithelium will enhance our understanding of the regulation of airway NO production.