Phorbol ester induces actin cytoskeleton rearrangements in cultured vascular smooth muscle cells. Calponin and SM22 α are major components of differentiated smooth muscle and potential regulators of actin cytoskeleton interactions. Here we show that actin fibers decorated with h1 CaP remain stable, whereas SM22 α-decorated actin bundles undergo rapid reorganization into podosomes within 30 min of PDBu exposure. Ectopic expression of GFP α-actinin had no effect on the stability of the actin cytoskeleton and α-actinin was transported rapidly into PDBu-induced podosomes. Our results demonstrate the involvement of CaP and SM22 α in coordinating the balance between stabilization and dynamics of the actin cytoskeleton in mammalian smooth muscle. We provide evidence for the existence of two functionally distinct actin filament populations and introduce a molecular mechanism for the stabilization of the actin cytoskeleton by the unique actin-binding interface formed by calponin family-specific CLIK 23 repeats.