The inflammatory cytokine, tumour necrosis factor-α (TNF), may be a key component of airway hyper-responsiveness producing an increased contractility. Our previous study has demonstrated that this increased contractility is the result of TNF-induced RhoA activation leading to an increased sensitivity to Ca²⁺ (Hunter et al, 2003). The aim of this study was to determine the intracellular mechanisms that result in TNF-induced RhoA activation in primary cultured human airway smooth muscle (ASM) cells (commercially obtained). RhoA activation was assayed using the GST-rhotekin ‘pull-down’ protocol (Hunter et al, 2003). Pretreatment of cells with 10 mM methyl β-cyclodextrin (Mβ-CD) (which depletes cholesterol thereby disrupting lipid raft domains) for 30 minutes resulted in a significant decrease in TNF-induced RhoA activation at 1 minute and 5 minutes stimulation (Mβ-CD treated 1.1 ± 0.2 fold increase, n=5; control 4.2 ± 0.4 fold increase, n=5, mean ± s.e.m., student’s t-test p<0.05). To determine if TNF receptors are present in lipid rafts, biotin-coupled TNF was used to label receptors, followed by incubation with a streptavidin-coupled fluorophore. TNF receptors were observed to co-localize predominantly with lipid raft regions of the plasma membrane in airway smooth muscle cells. Sucrose gradient fractionation of ASM cell extracts was carried out to separate Triton X-100 soluble and insoluble (lipid raft) fractions. Insoluble fractions, immunoblotted for proteins potentially involved in TNF-induced activation of RhoA, contained TNF receptor 1, several TNF receptor accessory proteins as well as RhoA. Caveolin-1, a marker of lipid rafts, was present only in raft fractions. Recruitment of TNF to lipid rafts following stimulation may be an important part of this signalling process. To assess if this occurs, cells were incubated with TNF and cell extracts separated into Triton X-100 soluble and insoluble (lipid raft) components. There was a significant increase in TNF receptor 1 recruitment to insoluble fractions following TNF stimulation for 1 minute (3.2 ± 0.4 fold increase compared to control, n=3). This was maintained after 5 minutes TNF stimulation. In conclusion, TNF-induced RhoA activation in human airway smooth muscle cells is dependent on functional lipid raft domains that contain components of TNF signalling, as well as RhoA. Following TNF stimulation, TNF receptors are recruited to the lipid rafts regions of the plasma membrane. Lipid rafts may therefore form an important platform for TNF-induced RhoA activation in ASM cells.