Membrane protein phosphorylation has been studied in resealed human erythrocyte ghosts by measuring the incorporation of 32P into spectrin and band 3. Norepinephrine- and Ca 2+-stimulated phosphate incorporation was diminished in ghosts depleted of calmodulin. Ghosts prepared with endogenous calmodulin showed Ca 2+- and norepinephrine-stimulated protein phosphorylation only when the ghosts had been resealed in the presence of [γ- 32P]ATP. Ghosts resealed with or without calmodulin in the presence of unlabeled ATP showed no net gain or loss of 32P when exposed to norepinephrine or a Ca 2+-specific ionophore. These observations suggest that Ca 2+ and norepinephrine stimulation of membrane protein phosphorylation is mediated by calmodulin-dependent spectrin kinase activity, and not by increased turnover of spectrin ATPase or by inhibition of phosphospectrin phosphatase.