The aim of the present study was to investigate the anti-inflammatory effects of 14,15-EET on reactivity and Ca²⁺ sensitivity in TNF-stimulated human bronchi. Tension measurements performed on either control, TNF or TNF + EET pre-treated bronchi revealed that 100 nM 14,15-EET pre-treatments significantly reduced the reactivity of TNF pre-treated tissues to contractile agonists. EET also normalized the relaxing response to isoproterenol in TNF-treated bronchi. Pretreatment with 100 nM 14,15-EET prevented TNF-induced IkBa degradation, as demonstrated by an increase in IkB protein levels on Western blot analysis. The anti-inflammatory properties of EET were mediated by the inhibition of IkB degradation, suggesting a lower activation of NFkB. The Ca²⁺ sensitivity of TNF-stimulated bronchi was also evaluated on B-escin-permeabilized preparations. Observed mean responses demonstrated that EET pretreatments abolished Ca²⁺ hypersensitivity developed by TNFa-stimulated bronchial explants. Moreover, 14,15-EET significantly reduced PDBu-induced Ca²⁺ sensitivity in TNF-stimulated bronchi. Western blot and RT-PCR analyses revealed that CPI-17 protein and transcript levels were increased in TNF treated bronchi, as opposed to being decreased in the presence of 14,15-EET. This eicosanoid also reduced U-46619-induced Ca²⁺ sensitivity, which is related to the activation of Rho-kinase pathway. These results were also correlated with an increase in protein staining and transcription level of p116^Rip, a RhoA inhibitory-binding protein. Altogether, these data demonstrate that 14,15-EET is a potent modulator of the hyper-reactivity triggered by TNF in human ASM cells.