The sarcoplasmic reticulum (SR) plays a key role in excitation-contraction coupling of skeletal muscle. The SR is composed of two continuous yet heterogeneous membrane compartments, the free or longitudinal SR (LSR) and cisternal SR. Cisternal SR is made up of free SR membrane, enriched in Ca2+-pump, and junctional SR (jSR) membrane, enriched in Ryanodine-sensitive Ca2+-release channel, and contains Calsequestrin within its lumen. Protein phosphorylation mediated by the Ca2+/calmodulin-dependent protein kinase II (CaMKII) has significant, distinct regulatory roles in both Ca2+ uptake and Ca2+ release. Kinase anchoring proteins (KAPs) constitute a novel mechanism for achieving cell compartmentalization of effectors in phosphorylation pathways. Here, targeting of aKAP, a CaMKII anchoring protein encoded within the a-CaMKII gene, was studied in transgenic skeletal muscle fibers of the adult rat soleus. The transgenes were epitope-tagged versions of aKAP and of a deletion mutant, allowing their specific immunodetection against the wild-type background. Our results show that aKAP is largely localized at the free SR and thus nearby the Ca2+-pump, a protein that can be modulated by CaMKII phosphorylation. Only minor colocalization was observed with the jSR ryanodine-sensitive Ca2+-release channel, which is a potential CaMKII target. In non-muscle cells, recombinant aKAP is targeted to endoplasmic reticulum (ER). Both ER and SR targeting requires the N-terminal hydrophobic region of aKAP. An unexpected additional specific localization that does not require the N-terminus was found in the nucleus, providing a first clue of how CaMKII can fulfill its nuclear functions in skeletal muscle.