Stress fibers play a crucial role in cell adhesion, motility, and morphogenesis, but the mechanisms of their assembly are not well understood. Using live cell microscopy, Pirta Hotulainen and Pekka Lappalainen found that stress fibers are generated through two distinct mechanisms. Dorsal stress fibers, connected to the substrate via focal adhesions, assemble through actin polymerization driven by formin (mDia1/DRF1) at these adhesions. In contrast, transverse arcs, which are not directly anchored to the substrate, form by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Interestingly, dorsal stress fibers and transverse arcs can convert into ventral stress fibers, which are anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that rapid association-dissociation kinetics of cross-linkers may be essential for their formation and contractility. Based on these findings, the authors propose a general model for the assembly and maintenance of contractile actin structures in cells.Stress fibers play a crucial role in cell adhesion, motility, and morphogenesis, but the mechanisms of their assembly are not well understood. Using live cell microscopy, Pirta Hotulainen and Pekka Lappalainen found that stress fibers are generated through two distinct mechanisms. Dorsal stress fibers, connected to the substrate via focal adhesions, assemble through actin polymerization driven by formin (mDia1/DRF1) at these adhesions. In contrast, transverse arcs, which are not directly anchored to the substrate, form by endwise annealing of myosin bundles and Arp2/3-nucleated actin bundles at the lamella. Interestingly, dorsal stress fibers and transverse arcs can convert into ventral stress fibers, which are anchored to focal adhesions at both ends. Fluorescence recovery after photobleaching analysis revealed that actin filament cross-linking in stress fibers is highly dynamic, suggesting that rapid association-dissociation kinetics of cross-linkers may be essential for their formation and contractility. Based on these findings, the authors propose a general model for the assembly and maintenance of contractile actin structures in cells.