Cofilin-actin bundles (rods), which form in axons and dendrites of stressed neurons, lead to synaptic dysfunction and may mediate cognitive deficits in dementias. Rods form abundantly in the cytoplasm of non-neuronal cells in response to many treatments that induce rods in neurons. Rods in cell lysates are not stable in detergents or with added calcium. Rods induced by ATP-depletion and released from cells by mechanical lysis were first isolated from two cell lines expressing chimeric actin-depolymerizing factor (ADF)/cofilin fluorescent proteins by differential and equilibrium sedimentation on OptiPrep gradients and then from neuronal and non-neuronal cells expressing only endogenous proteins. Rods contain ADF/cofilin and actin in a 1:1 ratio. Isolated rods are stable in dithiothreitol, EGTA, Ca(2+), and ATP. Cofilin-GFP-containing rods are stable in 500 mM NaCl, whereas rods formed from endogenous proteins are significantly less stable in high salt. Proteomic analysis of rods formed from endogenous proteins identified other potential components whose presence in rods was examined by immunofluorescence staining of cells. Only actin and ADF/cofilin are in rods during all phases of their formation; furthermore, the rapid assembly of rods in vitro from these purified proteins at physiological concentration shows that they are the only proteins necessary for rod formation. Cytoplasmic rod formation is inhibited by cytochalasin D and jasplakinolide. Time lapse imaging of rod formation shows abundant small needle-shaped rods that coalesce over time. Rod filament lengths measured by ultrastructural tomography ranged from 22 to 1480 nm. These results suggest rods form by assembly of cofilin-actin subunits, followed by self-association of ADF/cofilin-saturated F-actin.