Despite the high deposition of inositol hexakisphosphate (IP 6 ), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP 6 synthesis occurs through the sequential phosphorylation of I(1,4,5)P 3 by two gene products, Ipk2 and Ipk1, a IP 3 /IP 4 dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2␣ and AtIpk2 that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2␣ and AtIpk2 are 70% identical to each other and 12-18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2␣ and AtIpk2 have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P 3 to generate I(1,3,4,5,6)P 5 predominantly via an I(1,4,5,6)P 4 intermediate. While I(1,3,4,5)P 4 is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP 3 reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P 4 and I(1,2,3,4,6)P 5 , which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP 6 . Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP 4 and IP 5 production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class.Phosphorylation events are a vital part of intracellular communication and enable specific responses to various stimuli. As an asymmetric cyclitol harboring six hydroxyls, myo-inositol is a scaffold that has the potential to transduce a wealth of information through the combinatorial addition of phosphates.