Cultured myofibroblasts are characterized by stress fibers, containing α-smooth muscle actin (α-SMA) and by supermature focal adhesions (FAs), which are larger than FAs of α-SMA-negative fibroblasts. We have investigated the role of α-SMA for myofibroblast adhesion and FA maturation. Inverted centrifugation reveals two phases of initial myofibroblast attachment: during the first 2 h of plating microfilament bundles contain essentially cytoplasmic actin and myofibroblast adhesion is similar to that of α-SMA-negative fibroblasts. Then, myofibroblasts incorporate α-SMA in stress fibers, develop mature FAs and their adhesion capacity is significantly increased. When α-SMA expression is induced in 5 d culture by TGFβ or low serum levels, fibroblast adhesion is further increased correlating with a "supermaturation" of FAs. Treatment of myofibroblasts with α-SMA fusion peptide (SMA-FP), which inhibits α-SMA-mediated contractile activity, reduces their adhesion to the level of α-SMA negative fibroblasts. With the use of flexible micropatterned substrates and EGFP-constructs we show that SMA-FP application leads to a decrease of myofibroblast contraction, shortly followed by disassembly of paxillin- and β3 integrin-containing FAs; α5 integrin distribution is not affected. FRAP of β3 integrin-EGFP demonstrates an increase of FA protein turnover following SMA-FP treatment. We conclude that the formation and stability of supermature FAs depends on a high α-SMA-mediated contractile activity of myofibroblast stress fibers.