Rotational diffusion of band 3 proteins in human erythrocyte membranes is measured by observing flash-induced transient dichroism of the triplet probe, eosin-maleimide. At physiological temperature, both fast and slowly rotating populations of band 3 are present in the membrane. Rotational motion of band 3 is the same in membranes from young and old erythrocytes and is unchanged when the cholesterol:phospholipid mole ratio is varied from 1.34 to 1.66. Antibodies against glycophorin A immobilize band 3, indicating an association between these two integral membrane proteins. However, glycophorin A has little effect on the rotational motion of the complex, since band 3 rotation in En(a-) membranes (which lack glycophorin A) is similar to that observed in normal membranes. Cleavage of the cytoplasmic segment of band 3 by trypsin produces a considerable enhancement of band 3 rotational mobility. A similar effect is seen following extraction of bands 2.1 and 4.1 by sequential low salt-high salt treatment. It is concluded that up to 40% of band 3 has restricted rotational mobility due to interaction with the erythrocyte cytoskeleton.