A series of "P-labeled D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(l ,3,4,5)P41 analogues was enzymically prepared from the corresponding D-myo-inositol 1,4,5-trisphosphate [Ins( 1,4,5)P,] analogues using recombinant rat brain Ins(1 ,4,5)P3 3-kinase and [y-32P]ATP. Ins( l ,4,5)P, analogues with bulky groups at the 2-OH position, substitutions of phosphates by thiophosphates and D-6-deoxy-myo-Ins(l,4,5)P3 were tested. Using [3H]Ins(l ,4,5)P, and ATPyS, a [3H]Ins(l ,3,4,5)P4 analogue with a thiophosphate at the D-3 position was prepared. The D-4 andor D-5 phosphate group seemed to be important for 3-kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2-OH position did not prevent phosphorylation.The labeled Ins( 1 ,3,4,5)P4 analogues were purified and their degradation by type-I Ins( 1,4,5)P,l Ins(1 ,3,4,5)P4 5-phosphatase was compared with the degradation of Ins(1 ,3,4,5)P4. Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2-OH position did not prevent degradation. D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate could not be degraded by the 5-phosphatase, indicating the importance of the 6-OH group for 5-phosphatase action. D-6-Deoxy-myo-inositol 1,3,4,5-tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1 ,3,4,5)P4.The second messenger D-myo-inositol 1,4,5-trisphosphate [Ins( 1,4,5)P,] is generated by receptor-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP,) [l]. Its role in calcium mobilization from non-mitochondria1 stores is now generally accepted. In mammalian cells, the Ins(1,4,5)P, signal can be terminated by a 5-phosphatase to yield D-myo-inositol 1,4-diphosphate [2, 31, and also by a 3,4,5)P4 is metabolized by the same 5-phosphatase that dephosphorylates Ins(1,4,5)P3, and also by a 3-phosphatase [14].