The bulk of the red blood cell membrane proteins are partitioned between two multiprotein complexes, one associated with ankyrin R and the other with protein 4.1R. Here we examine the effect of phosphorylation of 4.1R on its interactions with its partners in the membrane. We show that activation of protein kinase C in the intact cell leads to phosphorylation of 4.1R at two sites, serine 312 and serine 331. This renders the 4.1R-associated transmembrane proteins GPC, Duffy, XK, and Kell readily extractable by nonionic detergent with no effect on the retention of band 3 and Rh, both of which also interact with 4.1R. In solution, phosphorlyation at either serine suppresses the capacity of 4.1R to bind to the cytoplasmic domains of GPC, Duffy, and XK. Phosphorylation also exerts an effect on the stability in situ of the ternary spectrin-actin-4.1R complex, which characterizes the junctions of the membrane skeletal network, as measured by the enhanced competitive entry of a β-spectrin peptide possessing both actin- and 4.1R-binding sites. Thus, phosphorylation weakens the affinity of 4.1R for β-spectrin. The two 4.1R phosphorylation sites lie in a domain flanked in the sequence by the spectrin- and actin-binding domain and a domain containing the binding sites for transmembrane proteins. It thus appears that phosphorylation of a regulatory domain in 4.1R results in structural changes transmitted to the functional interaction centers of the protein. We consider possible implications of our findings for the altered membrane function of normal reticulocytes and sickle red cells.