1 Bleomycin-induced lung injury is widely used as an experimental model to investigate the pathophysiology of pulmonary fibrosis but the alterations in the pharmacological responsiveness of airways isolated from bleomycin-exposed animals has been scarcely investigated. The aim of this study was to examine the in vitro tracheal responses to muscarinic receptor stimulation with carbachol in a rat bleomycin model. 2 Concentration-response curves to carbachol (10 nm to 0.1 mm) were obtained in tracheal rings isolated from Sprague-Dawley rats 14 days after endotracheal bleomycin or saline. The intracellular calcium signal in response to carbachol (10 microm) was measured by epifluorescence microscopy using fura-2 in primary cultures of tracheal smooth muscle cells from bleomycin- and saline-exposed rats. Circulating plasma tumour necrosis factor (TNF)-alpha/interleukin (IL)-1beta levels were measured by enzyme-linked immunosorbent assay. 3 Maximal contraction in response to carbachol was significantly greater in tracheal rings from bleomycin-exposed rats compared with controls (15.8 +/- 1.3 mN vs. 11.8 +/- 1.4 mN; n = 19, P < 0.05). 4 Carbachol (10 microm) elicited a transient increase of intracellular calcium with greater increment in tracheal smooth muscle cells from bleomycin-exposed rats compared with controls (372 +/- 42 nmvs. 176 +/- 20 nm; n = 7, P < 0.01). 5 Circulating plasma levels of TNF-alpha/IL-1beta were augmented in bleomycin-exposed rats compared with controls. Tissue incubation with TNF-alpha (100 ng ml(-1))/IL-1beta (10 ng ml(-1)) increased in vitro tracheal responsiveness to carbachol. 6 In conclusion, tracheal contraction in response to muscarinic receptor stimulation with carbachol was increased in bleomycin-exposed rats. This in vitro cholinergic hyperresponsiveness may be related to the augmented levels of inflammatory cytokines in bleomycin-exposed rats.