AbstractAll six isomericmyo‐inositol pentakisphosphates (InsP5), consisting of the twomesocompoundsmyo‐inositol 1,3,4,5,6‐pentakisphosphate [Ins(1,3,4,5,6)P5] (18) andmyo‐inositol 1,2,3,4,6‐pentakisphosphate [Ins(1,2,3,4,6)P5] (22) and two pairs of enantiomersmyo‐inositol 1,2,4,5,6‐pentakisphosphate [Ins(1,2,4,5,6)P5] (15)myo‐inositol 2,3,4,5,6‐pentakisphosphate [Ins(2,3,4,5,6)P5] (ent‐15) andmyo‐inositol 1,2,3,5,6‐pentakisphosphate [Ins(1,2,3,5,6)P5] (20)myo‐inositol 1,2,3,4,5‐pentakisphosphate [Ins(1,2,3,4,5)P5] (ent‐20), respectively, were synthesized. These compounds have been found in tissue, and although not resolved as pure enantiomers, their primary metabolism in a cytosolic extract from fetal calf thymus was therefore investigated by analytical HPLC. Four isomers were dephosphorylated to singly defined inositol tetrakisphosphates, while Ins(1,2,4,5,6)P5was phosphorylated tomyo‐inositol hexakisphosphate (InsP6). Interestingly, Ins(2,3,4,5,6)P5was the only isomer which was not metabolized. These data demonstrate that chemically synthesized, enantiomerically pure inositol pentakisphosphate isomers are valuable tools for the unravelling of the metabolic pathways o