The role of ankyrin in the formation and stabilization of the spectrin-based skeletal meshwork and of band 3 oligomers was studied by characterizing, in nb/nb mouse red cells, the effect of ankyrin deficiency on skeletal ultrastructure, band 3-skeleton associations, and band 3 oligomeric states. Despite severe ankyrin deficiency, nb/nb mouse red cell skeletal components formed a relatively uniform two-dimensional hexagonal array of junctional complexes cross-linked by spectrin tetramers. Treatment of nb/nb ghosts with the nonionic detergent C12E8 (octaethylene glycol n-dodecyl monoether) resulted in nearly complete extraction of band 3. The extracted band 3 was present exclusively as band 3 dimers. Fluorescence photobleaching recovery and polarized fluorescence depletion measurements showed increases in the laterally (33% vs 10%) and rotationally (90% vs 76%) mobile fractions of band 3 in intact nb/nb compared to control red cells. The rotational correlation time of the major fraction of band 3 molecules was 10-fold shorter in nb/nb compared to control red cells, indicating a significant relaxation of rotational constraints in nb/nb cells. These data suggest that, although ankyrin plays a major role in strengthening the attachment of the skeleton to the membrane bilayer, ankyrin is not required for the formation of a stable two-dimensional spectrin-based skeleton. The absence of band 3 tetramers in the membrane of ankyrin-deficient red cells suggests that ankyrin is required for the formation of stable band 3 tetramers.