The mechanical properties of human erythrocyte membrane are largely regulated by submembranous protein skeleton whose principal components are alpha- and beta-spectrin, actin, protein 4.1, adducin, and dematin. All of these proteins, except for actin, are phosphorylated by various kinases present in the erythrocyte. In vitro studies with purified skeletal proteins and various kinases has shown that while phosphorylation of these proteins can modify some of the binary and ternary protein interactions, it has no effect on certain other interactions between these proteins. Most importantly, at present there is no direct evidence that phosphorylation of skeletal protein(s) alters the function of the intact membrane. To explore this critical issue, we have developed experimental strategies to determine the functional consequences of phosphorylation of beta-spectrin on mechanical properties of intact erythrocyte membrane. We have been able to document that membrane mechanical stability is exquisitely regulated by phosphorylation of beta-spectrin by membrane-bound casein kinase I. Increased phosphorylation of beta-spectrin decreases membrane mechanical stability while decreased phosphorylation increases membrane mechanical stability. Our data for the first time demonstrate that phosphorylation of a skeletal protein in situ can modulate physiological function of native erythrocyte membrane.