A high molecular weight protein from the brush border of chicken intestinal epithelial cells has been purified. This protein (TW 260/240), a complex of two polypeptides with apparent molecular weights of 260,000 and 240,000, accounts for a significant amount of the terminal web organization. TW 260/240 is an F-actin-binding protein that also interacts with calmodulin. Rotary shadowing reveals long flexible rods of double-stranded morphology tightly connected at each end. TW 260/240 is quite distinct from smooth muscle filamin and macrophage actin-binding protein (APB), but, in spite of its higher contour length (265 nm), seems to be related to erythrocyte spectrin (194 nm for the tetramer). Immunofluorescence microscopy with antibodies against TW 260/240 indicates the existence of a submembranous organization distinctly different from that of stress fibers. We have compared TW 260/240 with fodrin, a brain protein known to occur in submembranous organization but not previously characterized in molecular terms. TW 260/240 and fodrin are clearly distinct molecules but are similar in many aspects. Ultrastructural, biochemical and immunological results indicate three distinct classes of rod-like high molecular weight actin-binding proteins, possibly reflected by the prototypes filamin (ABP), spectrin and TW 260/240 (fodrin). The latter group may be responsible for calmodulin control of submembranous microfilament structures in various nonmuscle cells.