Adenophostins A and B, which are metabolic products of the fungus Penicillium brevicompactum, are potent agonists at the D-myo-inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] receptor. In the current study, adenophostin A was approximately 50-fold more potent than Ins(1,4,5)P3 at both releasing Ca2+ from the intracellular stores of permeabilized platelets and displacing [3H]Ins(1,4,5)P3 from its receptor on rat cerebellar membranes. Various analogues bearing structural features found in the adenophostins and/or Ins(1, 4,5)P3 were examined to elucidate the molecular basis for the observed enhanced potency. 2-AMP did not induce Ca2+ release from permeabilized platelets or have any effect on Ins(1,4,5)P3-induced Ca2+ release. Two carbohydrate-based analogues, (2-hydroxyethyl)-alpha-D-glucopyranoside-2',3,4-trisphosphate and alpha,alpha'-trehalose-3,4,3',4'-tetrakisphosphate, could induce release of Ca2+ and displace [3H]Ins(1,4,5)P3 from its binding site on rat cerebellar membranes, although both were less potent than Ins(1,4,5)P3. In common with adenophostin A, release of Ca2+ from the intracellular stores could be inhibited by heparin, and both analogues were metabolically resistant. This study is the first to demonstrate the activity of a synthetic disaccharide at the Ins(1,4, 5)P3 receptor and that the Ins(1,4,5)P3 receptor is capable of accommodating an increased steric bulk. The minimal importance of the 2-hydroxyl group of Ins(1,4,5)P3 (occupied by the pyranoside oxygen in adenophostin) was confirmed by comparing the activity of DL-scyllo-Ins(1,2,4)P3 [which differs from Ins(1,4,5)P3 solely by the orientation of this hydroxyl group] with that of Ins(1,4,5)P3. An analogue of this compound, namely, DL-6-CH2OH-scyllo-Ins(1,2,4)P3, which possesses an equatorial hydroxymethyl group analogous to the 5'-hydroxymethyl group of adenophostin, was found to be equipotent to Ins(1,4,5)P3, demonstrating the tolerance of the Ins(1,4,5)P3 receptor to additional steric bulk at this position.