Calmodulin, an abundant protein in the red cell cytosol, exerts its effects on erythrocyte membrane properties via interactions with numerous proteins. To evaluate whether calmodulin might regulate association of protein 4.1 with one of its integral membrane protein anchors, protein 4.1 binding to inside-out erythrocyte membrane vesicles (IOVs) in the presence and absence of calmodulin and Ca2+ was examined. Ca2+ plus calmodulin was found to competitively inhibit protein 4.1 association with IOVs with a Ki of 1.4 microM and a maximal inhibition of 83%. In the absence of Ca2+, calmodulin still reduce protein 4.1 binding by 43%, consistent with the known Ca2+ independent association of calmodulin with protein 4.1. Ca2+ alone had no effect on protein 4.1-membrane interactions. Digestion studies revealed that both band 3 and glycophorin sites were similarly affected by calmodulin competition, suggesting all major protein 4.1 anchors are potentially regulated. In light of other data showing regulation of the same interactions by phosphoinositides, protein kinases, and the concentration of free cytosolic 2,3-diphosphoglycerate, it can be argued that association of protein 4.1 with integral protein anchors constitutes one of the more sensitively regulated interactions of the membrane.