Objective: Methotrexate (MTX) is very effective when used to treat chronic inflammatory diseases, and also induces apoptosis in nasal polyps (NPs). Increasing evidence suggests that Fas-Fas ligand (FasL) interactions activate multiple pathways involved in the regulation of immune and inflammatory cell functions. The aim of the present study was to identify pathways activated by Fas signaling when NPs were treated with MTX. Methods: Nasal polyps tissues were cultured using an air-liquid interface organ culture method. Cultures were maintained in the absence or presence of MTX (10 or 100 mu M) for 24 hours. The authors used the reverse transcription-polymerase chain reaction method and Western blotting to identify pathways activated by Fas when NPs were treated with MTX. Results: The Fas mRNA expression ratio was unchanged upon MTX treatment, but the FasL mRNA expression ratio was significantly higher in MTX-treated than nontreated polyps. In addition, the expression levels of the Fas and FasL proteins were significantly higher in polyps treated with both 10 and 100 mu M MTX compared with nontreated polyps. Conclusions: Methotrexate induces apoptosis in NPs via the Fas pathway. Future studies should explore the topical use of MTX for NP control. Methotrexate may be a useful alternative steroid-sparing agent for the treatment of NPs.