The cytokines transforming growth factor-β (TGF-β) and interleukin-13 (IL-13) have been implicated in the patho-physiology of asthma (Vignola et al., 1997; Batra et a., 2004) and may be contributing to the associated altered airway smooth muscle function. We hypothesized that these cytokines may be mediating this altered smooth muscle function through changes in RhoA/Rho-kinase signalling, which has also been implicated in allergen-induced airway hyper-responsiveness (Chiba et al., 2004). Human airway smooth muscle cells (hASMC) were obtained by bronchoscopy from moderate asthmatic or healthy subjects. We examined the protein expression of RhoA, Rho-kinase (ROCK), myosin phosphatase targeting subunit-1 (MYPT-1), myosin light-chain-20 (MLC20), and the Rho-specific guanine nucleotide exchange factor p115-RhoGEF in serum-starved healthy hASMC with or without prior 24 hr incubation with TGF-β (10ng/ml) or IL-13 (10ng/ml) or the two cytokines in combination. We also investigated differences in the protein expression of p115-RhoGEF in serum-starved hASMCs derived from moderate asthmatics compared to gender-matched healthy subjects (all male), and in lung tissue of ovalbumin (OVA) sensitized 1295SVJ/Black Swiss mice, compared to non-sensitized controls. Expression of MLC20, MYPT-1 and p115-RhoGEF in healthy hASMCs were all significantly enhanced by TGF-β and by TGF-β in combination with IL-13, but not by IL-13 alone, whereas expression of RhoA and ROCK were not altered by any of the treatments (Fig. 1; expressed as percentage of control vehicle treated cells; * P<0.05, **P<0.01, ***P<0.001; derived from one-way repeated measures ANOVA with Holm-Sidak post-tests for individual comparisons). Expression of p115-RhoGEF was approximately 3-fold greater both in asthmatic hASMC compared to controls and in OVA-sensitized mouse lung compared to controls (Fig. 2, *P<0.05 by unpaired t-test, n=3 in all cases, data expressed as a ratio of p115-RhoGEF/GAPDH expression). Our data suggest that TGF-β-induced changes in expression of RhoA-associated proteins may contribute to altered airway smooth muscle function in asthma. In particular, the guanine nucleotide exchange factor p115-RhoGEF, which activates RhoA in response to G-protein coupled receptor activation and integrin engagement (Wells et al., 2001; Dubash et al.,2007), may be important in mediating enhanced RhoA/Rho-kinase activity in asthmatic and allergen-sensitized airways.