A series of 32P-labeled D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3-kinase and [gamma-32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2-OH position, substitutions of phosphates by thiophosphates and D-6-deoxy-myo-Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATP gamma S, a [3H]Ins(1,3,4,5)P4 analogue with a thiophosphate at the D-3 position was prepared. The D-4 and/or 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)P3/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.