Recent studies have shown that inositol 1,4,5-trisphosphate 3-kinase isoform B (IP3KB) possesses important roles in the development of immune cells. IP3KB can be targeted to multiple cellular compartments, among them nuclear localization and binding in close proximity to the plasma membrane. The B isoform is the only IP3K that is almost ubiquitously expressed in mammalian cells. Detailed mechanisms of its targeting regulation will be important in understanding the role of Ins(1,4,5)P 3 phosphorylation on subcellular calcium signaling and compartment-specific initiation of pathways leading to regulatory active higher phosphorylated inositol phosphates. Here, we identified an exportin 1-dependent nuclear export signal ( 134 LQRELQNVQV) and characterized the amino acids responsible for nuclear localization of IP3KB ( 129 RKLR). These two targeting domains regulate the amount of nuclear IP3KB in cells. We also demonstrated that the localization of IP3KB at the plasma membrane is due to its binding to cortical actin structures. Intriguingly, all three of these targeting activities reside in one small polypeptide segment (amino acids 104 -165), which acts as a multitargeting domain (MTD). Finally, a hitherto unknown subnuclear localization of IP3KB could be demonstrated in rapidly growing H1299 cells. IP3KB is specifically enriched at nuclear invaginations extending perpendicular between the apical and basal surface of the nucleus of these flat cells. Such nuclear invaginations are known to be involved in Ins(1,4,5)P 3 -mediated Ca 2؉ signaling of the nucleus. Our findings indicate that IP3KB not only regulates cytoplasmic Ca 2؉ signals by phosphorylation of subplasmalemmal and cytoplasmic Ins(1,4,5)P 3 but may also be involved in modulating nuclear Ca 2؉ signals generated from these nuclear envelope invaginations.It is well established that the highly inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ) 2 -specific IP3Ks remove the Ca 2ϩ -releasing second messenger Ins(1,4,5)P 3 by phosphorylating it to Ins(1,3,4,5)P 4 (1) supporting the formation of higher phosphorylated inositols in the cell (2). So far, three isoforms of IP3K, termed A (3), B (4), and C (5), have been identified in mammals. Full-length cDNAs encoding IP3K isoform B (IP3KB) were cloned from man (6) and rat (7). IP3KB was mapped to the telomeric end of human chromosome 1 (8, 9) and identified as a candidate gene in the pathogenesis of multiple sclerosis (10), Alzheimer disease (11), and malignant melanoma (12). But most importantly, IP3KB is a promising new target in immune disorders (13). Transgenic disruption of the IP3KB gene in mice causes a significant decrease in Ins(1,3,4,5)P 4 levels in thymocytes leading to a block of positive selection and consequently to a severe T cell deficiency (14). Recent studies described additional functions of IP3KB in selection and survival of B cells (15, 16), signaling of neutrophils (17), and modulation of myelopoiesis (18). In mammalian cells, multiple intracellular localizations of IP3KB, namely to the plasma membr...