We combine total internal reflection fluorescence microscopy with continuous photobleaching to study the growth mode of focal adhesions in rat embryonic fibroblasts (REF-52). We measured GFP-labelled beta 3 integrin, which exhibits a diffusion rate of 10(-14)-10(-13) m(2)/s when it is not bound to a focal adhesion. We show that exchange of beta 3 integrin between focal adhesions and the surrounding membrane occurs only at the edges of the focal adhesion. We, therefore, conclude that focal adhesions show an edge-on/edge-off growth, in contrast to the bulk-on/bulk-off growth mode that is assumed in many theoretical treatments. (C) Koninklijke Brill NV, Leiden, 2010