Ins(1,4,5) P 3 3-kinase (IP3K) phosphorylates the Ca 2+ -mobilizing second messenger Ins(1,4,5) P 3 to yield the putative second messenger Ins(1,3,4,5) P 4 . A HeLa cell line was established expressing the rat B isoform of IP3K under the control of an inducible promoter. The IP3KB-transfected cell line possessed 23-fold greater IP3K activity than untransfected cells after induction of IP3KB expression, but only 0.23-fold greater activity when IP3KB expression was not induced. Elevating IP3KB expression significantly reduced levels of Ins(1,4,5) P 3 and increased levels of Ins(1,3,4,5) P 4 after stimulation of cells with histamine, but had no effect on basal levels. Histamine- and ATP-evoked cytosolic Ca 2+ responses were dramatically reduced upon elevation of IP3KB expression. On stimulation with a supramaximal dose of histamine, 67% of cells induced to express IP3KB gave no detectable elevation in cytosolic Ca 2+ , compared with 3% of uninduced cells. The quantity of Ca 2+ within thapsigargin-sensitive and -insensitive stores was unaffected by elevation of IP3KB expression, as was capacitative Ca 2+ entry. These data suggest that IP3KB may play a significant role in the regulation of Ins(1,4,5) P 3 levels, and consequently in Ca 2+ responses following stimulation of cells with Ins(1,4,5) P 3 -elevating agonists.