Binding activity of [ 3H]inositol 1,3,4,5-tetrakisphosphate (InsP 4) was characterized with rat cerebellar membranes. Two types of InsP 4 analog with either the aminobenzoyl or the aminocyclohexanecarbonyl group on the 2nd position of InsP 4 have been synthesized and their effects on the binding activity were also examined. [ 3H]InsP 4 binding was gradually displaced by increasing amounts of unlabeled InsP 4, with an IC 50 of 60–170 nM, depending on the pH values. The binding was sharply increased at acidic pH and millimolar concentrations of Ca 2+, this being in clear contrast with [ 3H]InsP 3 binding noted in the same species of tissue. Heparin inhibited the binding, with an IC 50 of 1.7, 3 or 20 μg/ml at pH 8.3, 7.2 or 5.0, respectively. Adenine nucleotide inhibited the binding more potently than did [ 3H]InsP 3 binding. InsP 4 analogs were as effective as InsP 4 in displacing [ 3H]InsP 4 from rat cerebellar membranes, thereby indicating that the 2nd hydroxl group may not be involved in recognition of InsP 4 by its binding sites.