Ca2+ accumulated by rat cerebellum microsomes in the presence of MgATP was released by added inositol tetrakisphosphate [Ins(1,3,4,5)P4]. The concentrations of D-myo-inositol-1,4,5-trisphosphate [D-Ins(1,4,5)P3], D-Ins(1,3,4,5)P4, and DL-Ins(1,3,4,5)P4 required for half-maximal release were 0.15, 4.6, and 7.5 microM, respectively. Maximal concentrations of InsP4 released only 70% of the Ca2+ released by maximal concentrations of Ins(1,4,5)P3. Inositol pentakisphosphate and D-myo-inositol-1,3,4-trisphosphate were relatively inactive. Additional Ca2+ was released when Ins(1,4,5)P3 (or a nonhydrolyzable analog) was added after completion of InsP4-mediated Ca2+ release but not when this sequence of additions was reversed. This indicates that InsP4 releases Ca2+ from part of the InsP3-releasable compartment. No evidence for synergism between InsP4 and InsP3 was obtained and responses to suboptimal concentrations of both inositol phosphates were approximately additive. Heparin was a potent inhibitor of InsP4-mediated Ca2+ release. Inhibition by heparin was competitive with respect to InsP4 concentration and the Ki for heparin was 0.6 microgram/ml (approximately 100 nM), irrespective of whether InsP4 or InsP3 was used as an agonist. A 3-phosphatase capable of converting [3H]Ins(1,3,4,5)P3 to [3H]Ins(1,4,5)P3 could not be detected in cerebellum microsomes. Hence, we conclude that, in vitro, receptors capable of recognizing Ins(1,3,4,5)P4 can also be coupled directly to a Ca2+ release system.