To explore the biological significance of the D-3 position phosphorylation of the second messenger D-myo-inositol 1,4,5-trisphosphate (IP3) in cellular signalling, three novel D-3-substituted analogues of IP3 have been synthesized; their binding and Ca2+-release profiles at the IP3-receptor have been studied and shown to correlate with the steric requirement of the D-3 substituent.