We present evidence that inositol 1,3,4,5,6-pentakisphosphate acts as an antagonist of inositol triphosphate receptors in vitro by dampening the activity of Ca(2+)-mobilizing inositol phosphates. This antagonism bears physiological significance as cells maintain substantial quantities of this phosphoinositol in the cytosol, especially during cell differentiation or proliferation. Over a concentration range from 30 to 150 microM, this pentakisphosphate causes a two- to five-fold increase in the EC50 values of inositol 1,4,5-trisphosphate and other active phosphoinositols in effecting Ca2+ efflux from brain microsomes. This specific inhibition results from competitive receptor binding, and is not noted with inositol hexaphosphate or related metabolites at comparable concentrations. Thus, inositol 1,3,4,5,6-pentakisphosphate in concert with other allosteric modulators may constitute an integral component for the receptor regulation.