The purpose of this study was to analyze where monomeric actin first becomes incorporated into the sarcomeric units of the stress fibers. We microinjected fluorescently labeled actin monomers into two cell lines that differ in the sarcomeric spacings of alpha-actinin and nonmuscle myosin II along their stress fibers: REF-52, a fibroblast cell line, and PtK2, an epithelial cell line. The cells were fixed at selected times after microinjection (30 s and longer) and then stained with an alpha-actinin antibody. Localization of the labeled actin and alpha-actinin antibody were recorded with low level light cameras. In both cell types, the initial sites of incorporation were in focal contacts, lamellipodia and in punctate regions of the stress fibers that corresponded to the alpha-actinin rich dense bodies. The adherent junctions between the epithelial PtK2 cells were also initial sites of incorporation. At longer times of incorporation, the actin fluorescence extended along the stress fibers and became almost uniform. We saw no difference in the pattern of incorporation in peripheral and perinuclear regions of the stress fibers. We propose that rapid incorporation of monomeric actin occurs at the cellular sites where the barbed ends of actin filaments are concentrated: at the edges of lamellipodia, the adherens junctions, the attachment plaques and in the dense bodies that mark out the sarcomeric subunits of the stress fibers.