This study investigated the effects of airway inflammation elicited by intraperitoneal and intratracheal lipopolysaccharide administration to guinea pigs on the activity of tracheal epithelium-derived relaxant factor (EpDRF). Acetylcholine induced epithelium-dependent relaxation in precontracted rat anococygeus muscle placed in guinea pig trachea (coaxial bioassay). Indomethacin, N-nitro-l-arginine methyl ester, aminoguanidine and l-canavanine did not alter this relaxation excluding the role of prostaglandins and nitric oxide (NO). Intraperitoneal lipopolysaccharide potentiated the acetylcholine response, which was reversed by aminoguanidine and l-canavanine while intratracheal lipopolysaccharide inhibited acetylcholine-induced relaxation. Lipopolysaccharide pretreatments did not cause epithelial damage but induced inflammatory cell infiltration. These results suggested that systemic lipopolysaccharide administration did not alter the EpDRF response but resulted in NO synthase induction, thus NO participated in relaxation to acetylcholine in coaxial bioassay system. On the other hand, airway inflammation induced by intratracheal lipopolysaccharide attenuated the synthesis/release of EpDRF without altering the epithelium morphology.