The present study investigated the characteristics of D- myo-inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3) binding sites in crude membrane preparations of rabbit aortic smooth muscle. A particular aim was to demonstrate if increases in cytoplasmic cyclic guanosine 3′:5′ monophosphate (cGMP), which mediates the effect of nitrovasodilators, may cause smooth muscle relaxation in part by the displacement of Ins(1,4,5)P 3 binding. Negligible Ins(1,4,5)P 3 binding was observed at pH < 7, while maximum binding occurred over the pH range 8–9. Saturation analysis of isotopic dilution binding data revealed an apparently homogenous population of Ins(1,4,5)P 3 binding sites with a K D of 4.02 ± 0.53 nM and a B max of 27.7 ± 4.6 fmol/mg protein. Heparin, an Ins(1,4,5)P 3 receptor antagonist, inhibited binding with an IC 50 of 11.43 ± 2.81 ωg/ml. The ability of other polyphosphate compounds to inhibit Ins(1,4,5)P 3 binding in this preparation was also examined. D- myo-Inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P 4), adenosine 5′-triphosphate (ATP) and guanosine 5′-triphosphate (GTP) inhibited Ins(1,4,5)P 3 binding, although each was significantly less potent that Ins(1,4,5)P 3. In contrast, cyclic guanosine 3′:5′ monophosphate (cGMP) did not significantly alter Ins(1,4,5)P 3 binding in rabbit aortic smooth muscle. This observation suggests that competitive inhibition of Ins(1,4,5)P 3 receptor binding is not an important consideration in cGMP-mediate vascular smooth muscle cell relaxation.