Insulin-secreting pancreatic islet β-cells express a Group VIA Ca 2+-independent phospholipase A2 (iPLA2β) that contains a calmodulin binding site and protein interaction domains. We identified Ca2+/calmodulin-dependent protein kinase IIβ (CaMKIIβ) as a potential iPLA2β-interacting protein by yeast two-hybrid screening of a cDNA library using iPLA2β cDNA as bait. Cloning CaMKIIβ cDNA from a rat islet library revealed that one dominant CaMKIIβ isoform mRNA is expressed by adult islets and is not observed in brain or neonatal islets and that there is high conservation of the isoform expressed by rat and human β-cells. Binary two-hybrid assays using DNA encoding this isoform as bait and iPLA2β DNA as prey confirmed interaction of the enzymes, as did assays with CaMKIIβ as prey and iPLA2β bait. His-tagged CaMKIIβ immobilized on metal affinity matrices bound iPLA2β, and this did not require exogenous calmodulin and was not prevented by a calmodulin antagonist or the Ca2+ chelator EGTA. Activities of both enzymes increased upon their association, and iPLA2β reaction products reduced CaMKIIβ activity. Both the iPLA2β inhibitor bromoenol lactone and the CaMKIIβ inhibitor KN93 reduced arachidonate release from INS-1 insulinoma cells, and both inhibit insulin secretion. CaMKIIβ and iPLA 2β can be coimmunoprecipitated from INS-1 cells, and forskolin, which amplifies glucose-induced insulin secretion, increases the abundance of the immunoprecipitatable complex. These findings suggest that iPLA 2β and CaMKIIβ form a signaling complex in β-cells, consistent with reports that both enzymes participate in insulin secretion and that their expression is coinduced upon differentiation of pancreatic progenitor to endocrine progenitor cells. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.