Synthetic DL-myo-inositol 1,2,4,5-tetrakisphosphate (DL-Ins-(1,2,4,5)P4) functioned as a full agonist, with only 3-fold less potency than D-Ins(1,4,5)P3 in eliciting the release of Ca2+ from nonmitochondrial pools of permeabilized rat basophilic leukemic cells. DL-Ins(1,2,4,5)P4 inhibited the binding of D-[3H]Ins(1,4,5)P3 to the purified D-Ins(1,4,5)P3 receptor with almost the same potency as seen for the Ca2+ release. This compound inhibited the hydrolysis of D-[3H]Ins(1,4,5)P3 to D-[3H]Ins(1,4)P2 catalyzed by erythrocyte ghosts, with a Ki value of as low as 1.4 microM, but it could not serve as a substrate for the same enzyme. D-Ins(1,4,5)P3 3-kinase in rat brain cytosol did not recognize the compound at concentrations up to 30 microM. Thus, it would appear that DL-Ins(1,2,4,5)P4 can serve as a potent and long lasting experimental and pharmacological tool for stimulating D-Ins(1,4,5)P3-mediating processes.