Nitric oxide is an important mediator of inflammatory responses in the lung and a key regulator of pulmonary vascular and bronchomotor tone. We have shown that the inducible nitric oxide synthase (iNOS) isoform is continuously expressed in human airway epithelium at mRNA and protein/activity levels in vivo. However, removal of epithelial cells from the in vivo airway environment resulted in rapid loss of iNOS expression, which suggested that expression is dependent upon conditions and/or factors present in the airway. To investigate the mechanisms responsible for maintenance of expression in vivo, we evaluated regulation of iNOS expression in primary human airway epithelial cells. Interferon-gamma (IFN-gamma) was sufficient for induction of iNOS in primary human airway epithelial cells (HAEC) in vitro, and interleukin-4 (IL-4) potentiated the expression through stabilization of iNOS mRNA. The IFN-gamma/IL-4-induced iNOS expression in HAEC was delayed in onset and prolonged with expression up to 1 week. Furthermore, transfer of overlying culture media [conditioned media (CM)] to other HAEC led to iNOS induction. Interestingly, IFN-gamma/IL-4 induction of iNOS was dependent on new protein synthesis, whereas CM induction of iNOS was not. IFN-gamma and IL-4 activated signal transducers and activators of transcription (STAT1 and STAT6) in HAEC, but CM transfer to HAEC produced even higher levels of STAT1 activation than achieved by direct addition of cytokines. Thus, IFN-gamma/IL-4, which occurs in human lung lining fluid, led to iNOS expression in human airway epithelium through production of soluble mediators and stabilization of mRNA.