Costameres are mechano-sensory sites of focal adhesion in the sarcolemma that reinforce the muscle-fiber composite and provide an anchor for myofibrillogenesis. We hypothesized that elevated content of the integrin-associated regulator of costamere turnover in culture, focal adhesion kinase (FAK), drives changes in costamere component content in anti-gravity muscle in a load-dependent way in correspondence with altered muscle weight. The content of FAK in soleus muscle being phosphorylated at auto-regulatory tyrosine 397 (FAK-pY397) was increased after 20 seconds of stretch. FAK-pY397 content remained elevated after 24 hours of stretch-overload due to up-regulated FAK content. Overexpression of FAK in soleus muscle fibers by means of gene electro-transfer increased the beta 1 integrin (+56%) and meta-vinculin (+88%) content. Alpha 7 integrin (p=0.46) and gamma-vinculin (p=0.18) content was not altered after FAK overexpression. Co-overexpression of the FAK inhibitor FRNK reduced FAK-pY397 content by 33% and increased the percentage of fast type fibers that arose in connection with hybrid fibers with gene transfer. Transplantation experiments confirmed the association of FRNK expression with slow-to-fast fiber transformation. 7 days of unloading blunted the elevation of FAK-pY397, beta 1 integrin and meta-vinculin content with FAK-overexpression and this was reversed by one day of reloading. The results highlight that the expression of components for costameric attachment sites of myofibrils is under load- and fiber type-related control via FAK and its inhibitor FRNK.