Phosphatidylinositol (PI) kinases are key molecules that participate in the phosphoinositide signaling in the cytoplasm. Despite the accumulating evidence that supports the existence and operation of independent PI signaling system in the nucleus, the exact location of the PI kinases inside the nucleus is not well defined. Here we show that PI4-kinases IIα and IIβ, which play central roles in PI(4,5)P(2) synthesis and PI signaling, are localized in numerous small nucleoplasmic vesicles that function as inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3))-sensitive Ca(2+) stores. This is in accord with the past results that showed the localization of PI4(P)5-kinases that are essential in PI(4,5)P(2) production and PI(4,5)P(2) in nuclear matrix. Along with PI(4,5)P(2) that also exists on the nucleoplasmic vesicle membranes, the localization of PI4-kinases IIα and IIβ in the nucleoplasmic vesicles strongly implicates the vesicles to the PI signaling as well as the Ins(1,4,5)P(3)-depenent Ca(2+) signaling in the nucleus. Accordingly, the nucleoplasmic vesicles indeed release Ca(2+) rapidly in response to Ins(1,4,5)P(3). Further, the Ins(1,4,5)P(3)-induced Ca(2+) release studies suggest that PI4KIIα and IIβ are localized near the Ins(1,4,5)P(3) receptor (Ins(1,4,5)P(3)R)/Ca(2+) channels on the Ca(2+) store vesicle membranes. In view of the widespread presence of the Ins(1,4,5)P(3)-dependent Ca(2+) store vesicles and the need to fine-control the nuclear Ca(2+) concentrations at multiple sites along the chromatin fibers in the nucleus, the existence of the key PI enzymes in the Ins(1,4,5)P(3)-dependent nucleoplasmic Ca(2+) store vesicles appears to be in perfect harmony with the physiological roles of the PI kinases in the nucleus.