The focal adhesion complexes (or costameres) are macromolecular assemblies connecting the extracellular matrix to the cytoplasmic cytoskeleton. Focal adhesion kinase (FAK) is one such costameric component exhibiting important structural and non-receptor tyrosine kinase activities in response to integrin engagement and growth factor stimulation. Exemplifying its importance, overexpression of FAK stimulates muscle hypertrophy in vivo (1) and its expression is reciprocal to loading patterns (i.e. downregulated in atrophy/ upregulated in hypertrophy (2)). On this basis, we reasoned that reducing FAK expression by short hairpin (sh)RNA interference would restrict cell growth associated with insulin-like growth factor-1 (IGF-1) and contraction (mechanotransduction). Stably transfected C2C12 cells harbouring FAK targeted (pLKO.1-mFAK) or scrambled shRNA were developed using lentiviral transfection techniques (3). FAK and scrambled shRNA myotubes (4-5 d after differentiation) were incubated for 72 h with IGF-1 Long R3 (10 ng.ml-1 replenished daily) for measurement of total protein (µg/well by dissolution in 0.3 M NaOH). Immunoblotting was used to determine FAK levels and phosphorylation of ‘hypertrophy’ signalling targets (i.e. Akt-mammalian target of rapamycin (mTOR)) after 1 h IGF-1 incubation. In separate experiments, FAK and scrambled shRNA myotubes were contracted for 24 h (0.2 Hz, 5 V, 2 ms) using a C-pace system, before assay of total protein. Results were analysed by one-way ANOVA and Tukey’s post-hoc testing with the level of significance set at P<0.05. Data are presented as mean percentage differences ± standard error. FAK depletion was confirmed at the protein level (-90%: FAK vs. scrambled shRNA; P<0.00001). While IGF-1 treatment for 72 h elicited robust increases in total protein in scramble shRNA cells (+77±10%; P<0.001), this was markedly blunted in FAK shRNA cells (+34±14%; P=0.14). In response to 1 h IGF-1 treatment of scrambled shRNA cells, phosphorylated proteins of the Akt-mTOR pathway increased: p-Akt (Ser473: +180±9%; P<0.001), p-mTOR (Ser2448: +120±5%; P<0.001) and p-GSK3β (Ser9/21: 70±9%; P<0.001). However, in FAK shRNA cells, increases in phosphorylation were significantly attenuated at 1 h: (p-Akt, -31%; p-mTOR, -32%; p-GSK3β, -30% vs. IGF-1-treated scramble shRNA cells; all P<0.05). In contrast, IGF-1-induced increases in p-p70S6K (P<0.05) were not different (FAK shRNA Thr389: +41±13%; scrambled shRNA: +46±10%; P<0.01). Despite FAK shRNA cells displaying a contractile phenotype, increases in total protein in scrambled shRNA cells after 24 h contraction (+57±9%; P<0.01) were absent in FAK shRNA cells (+7±11% P=0.65). We have identified that FAK represents a key component of growth factor (IGF-1) and contraction (mechanotransduction)-mediated cell growth, possibly via modulation of Akt-mTOR-signalling.