Cortexillins I and II of D. discoideum constitute a novel subfamily of proteins with actin-binding sites of the alpha-actinin/spectrin type. The C-terminal halves of these dimeric proteins contain a heptad repeat domain by which the two subunits are joined to form a two-stranded, parallel coiled coil, giving rise to a 19 nm tail. The N-terminal domains that encompass a consensus actin-binding sequence are folded into globular heads. Cortexillin-linked actin filaments form preferentially anti-parallel bundles that associate into meshworks. Both cortexillins are enriched in the cortex of locomoting cells, primarily at the anterior and posterior ends. Elimination of the two isoforms by gene disruption gives rise to large, flattened cells with rugged boundaries, portions of which are often connected by thin cytoplasmic bridges. The double-mutant cells are multinucleate owing to a severe impairment of cytokinesis.