αKAP is an anchoring protein for the Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and is encoded within the same gene as the CaMKIIα isoform. αKAP co-assembles with CaMKII and targets such heteromers to the membrane of the sarcoplasmic reticulum, where CaMKII can regulate Ca 2+ homeostasis. CaMKII has also nuclear functions in skeletal muscle, however, the nuclear targeting mechanism has been elusive. We show here that developmentally regulated splicing of exon Eα B generates a functional nuclear localization signal (NLS) in αKAP B , the dominant αKAP variant in mature muscle. The αKAP A variant lacks the NLS and dominates in developing muscle before and around birth. Both αKAP variants localize to membranes, but a small fraction of αKAP B is additionally found in the nucleus. Indeed, α-karyopherins that mediate nuclear import bound to αKAP B but not αKAP A in vitro. When the N-terminal membrane anchor of αKAP was deleted, localization of αKAP B but not αKAP A became predominantly nuclear. Co-expression of constitutively active CaMKI and IV, which do not bind to αKAP, interfered with nuclear localization of αKAP B . CaMKIIα was found essentially exclusively in the cytoplasm when expressed in cell lines but was targeted to the nucleus when co-expressed with the nuclear form of αKAP B . Thus, nuclear targeting of cytoplasmic CaMKII isoforms by αKAP may be regulated by developmentally controlled alternative splicing and by protein kinases.