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 AtIpk2alpha and AtIpk2beta that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2alpha and AtIpk2beta are 70% identical to each other and 12-18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2alpha and AtIpk2beta 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.