TGFß is a widely distributed, pleiotropic cytokine implicated in tissue remodelling in many diseases including severe asthma. It is secreted in a latent form that requires activation for function. Mechanotransduction of intracellular forces through cell surface integrins can lead to TGFß activation. We have previously shown that human airway smooth muscle (HASM) cells can activate TGFβ via αvβ5 integrins. In the present study we have investigated the effect of extracellular matrix (ECM) on basal TGFβ activation in primary asthmatic and non-asthmatic HASM cells. We assessed basal TGFβ activation in non-asthmatic (n=9) and asthmatic (n=7) HASM cells. TGFβ activation was assessed using a TGFβ reporter cell assay and a phosphorylated Smad2 ELISA. Cell contractility was assessed using a collagen gel contraction assay and traction force microscopy. ECM was isolated from HASM cells and cross-over experiments performed where non-asthmatic cells were cultured on asthmatic ECM and vice-versa then TGFβ activity determined. Expression of ECM crosslinking enzymes was determined at the mRNA and protein levels. Finally, expression of LOXL2 was determined in an Aspergillus fumigatus model of asthma.Asthmatic HASM cells activated 3-fold higher levels of TGFβ basally than non-asthmatic cells (p<0.01). Both diseased and control HASM cells increased TGFβ activation in response to methacholine confirming our previous data (Tatler et al 2011). A collagen gel contraction assay demonstrated that asthmatic HASM cells were hypercontractile compared with non-asthmatic cells under basal conditions (p<0.05) and that contractility weakly correlated with amount of TGFβ activated (p<0.05). Importantly, culturing non-asthmatic HASM cells on asthmatic ECM led to increased TGFβ activation (p<0.05) and culturing asthmatic HASM cells on non-asthmatic ECM decreased TGFβ activation (p<0.05). mRNA Expression of the ECM crosslinking enzymes LOXL2 and LOXL3 was significantly increased in asthmatic HASMs (p<0.05). Finally, LOXL2 protein was increased in asthmatic HASMs cells, and increased in the airway smooth muscle layer of animals challenged repeatedly with Asp. f compared with control challenged animals. In conclusion HASM cells derived from asthmatic patients exhibit enhanced activation of TGFβ compared with non-asthmatic HASM cells. This may be driven by the diseased ECM since asthmatic HASMs cells exhibit aberrant expression of ECM crosslinking enzymes.