Ca 2+/calmodulin-dependent protein kinase II (CaM kinase II) is present in a membrane-bound form that phosphorylates synapsin I on neuronal synaptic vesicles and the ryanodine receptor at skeletal muscle sarcoplasmic reticulum (SR), but it is unclear how this soluble enzyme is targeted to membranes. We demonstrate that αKAP, a non-kinase protein encoded by a gene within the gene of α-CaM kinase II, can target the CaM kinase II holoenzyme to the SR membrane. Our results indicate that αKAP (i) is anchored to the membrane via its N-terminal hydrophobic domain,(ii) can co-assemble with catalytically competent CaM kinase II isoforms and target them to the membrane regardless of their state of activation, and (iii) is co-localized and associated with rat skeletal muscle CaM kinase II in vivo. αKAP is therefore the first demonstrated anchoring protein for CaM kinase II. CaM kinase II assembled with αKAP retains normal enzymatic activity and the ability to become Ca 2+-independent following autophosphorylation. A new variant of β-CaM kinase II, termed β M-CaM kinase II, is one of the predominant CaM kinase II isoforms associated with αKAP in skeletal muscle SR.