The effects of tumour necrosis factor‐α (TNF) on guinea‐pig bronchial smooth muscle contractility were investigated. The Ca2+‐activated contractile response of permeabilized bronchial smooth muscle strips was significantly increased after incubation with 1 μg ml−1 TNF for 45 min. This TNF‐induced effect was not due to a further increase in intracellular Ca2+. The TNF‐induced Ca2+ sensitization was, at least partly, the result of an increase in myosin light chain20 phosphorylation. The intracellular signalling pathway involved in this effect of TNF was further investigated. Sphingomyelinase, a potential mediator of TNF, had no effect on Ca2+ sensitivity of permeabilized bronchial smooth muscle. Also, p42/p44 mitogen‐activated protein kinase (p42/p44mapk), activated by TNF in some cell types, did not show an increased activation in bronchial smooth muscle after TNF treatment. In conclusion, TNF may activate a novel signalling pathway in guinea‐pig bronchial smooth muscle leading to an increase in myosin light chain20 phosphorylation and a subsequent increase in Ca2+ sensitivity of the myofilaments. This pathway does not appear to involve sphingomyelinase‐liberated ceramides or activation of p42/p44mapk. Given the importance of TNF in asthma, this TNF‐induced Ca2+ sensitization of the myofilaments may represent a mechanism responsible for airway hyper‐responsiveness.