One prominent cytoskeletal feature of non-mammalian vertebrate erythrocytes is the marginal band (MB), composed of microtubules. However, there have been several reports of MB-associated F-actin. We have further investigated the function of MB-associated F-actin, using newt erythrocytes having large, thick MBs. Confocal microscopy revealed a distinctive band of F-actin colocalizing point- by-point with MB microtubules. Furthermore, the F-actin band was present in isolated elliptical MBs, but absent in membrane skeletons lacking MBs. F-actin depolymerizing agents did not affect F-actin band integrity in isolated MBs, indicating its non-dynamic state. However, exposure to elastase resulted in F-actin removal and MB circularization. These results provide evidence of a strong association of F-actin with MB microtubules in mature ellipsoidal erythrocytes. To assess the true extent of mechanical stress on the cytoskeleton, erythrocytes were observed by video microscopy during flow in vivo. Moving with long axis parallel to flow direction, cells underwent reversible shape distortion as they collided vigorously with other erythrocytes and vessel walls. In addition, cells twisted into figure-8 shapes, a cytoskeletal property that may provide physiological advantages during flow. Our results, together with those of others, yield a consistent picture in which developing erythrocytes undergo transition from spheroids to immature discoids to mature ellipsoids. The causal step in discoid formation is biogenesis of circular MBs with sufficient flexural rigidity to determine cell shape. F-actin binding to MB microtubules then creates a composite system, enhancing flexural rigidity to produce and maintain ellipsoidal shape during the physical challenges of blood flow in vivo.